Rate Reactive Oxygen Species Research Articles

Quality Saving Mechanisms of Mitochondria during Aging in a Fully Time-Dependent Computational Biophysical Model.

Mitochondria are essential for the energy production of eukaryotic cells. During aging mitochondria run through various processes which change their quality in terms of activity, health and metabolic supply. In recent years, many of these processes such as fission and fusion of mitochondria, mitophagy, mitochondrial biogenesis and energy consumption have been subject of research. Based on numerous experimental insights, it was possible to qualify mitochondrial behaviour in computational simulations. Here, we present a new biophysical model based on the approach of Figge et al. in 2012. We introduce exponential decay and growth laws for each mitochondrial process to derive its time-dependent probability during the aging of cells. All mitochondrial processes of the original model are mathematically and biophysically redefined and additional processes are implemented: Mitochondrial fission and fusion is separated into a metabolic outer-membrane part and a protein-related inner-membrane part, a quality-dependent threshold for mitophagy and mitochondrial biogenesis is introduced and processes for activity-dependent internal oxidative stress as well as mitochondrial repair mechanisms are newly included. Our findings reveal a decrease of mitochondrial quality and a fragmentation of the mitochondrial network during aging. Additionally, the model discloses a quality increasing mechanism due to the interplay of the mitophagy and biogenesis cycle and the fission and fusion cycle of mitochondria. It is revealed that decreased mitochondrial repair can be a quality saving process in aged cells. Furthermore, the model finds strategies to sustain the quality of the mitochondrial network in cells with high production rates of reactive oxygen species due to large energy demands. Hence, the model adds new insights to biophysical mechanisms of mitochondrial aging and provides novel understandings of the interdependency of mitochondrial processes.

The effect of fenofibrate on expression of genes involved in fatty acids beta-oxidation and associated free-radical processes

Fenofibrate is a synthetic ligand for peroxisome proliferator-activated receptors subtype alpha (PPARa); it is used for the treatment of a wide range of metabolic diseases such as hypertriglyceridemia, dyslipidemia, diabetes and various neurodegenerative diseases. We have studied the effect of fenofibrate on b-oxidation of fatty acids and related free-radical processes. The most effective concentration of fenofibrate (0.3%) added to the chow caused a significant decrease of the body weight of mice. The data obtained by quantitative PCR demonstrated increased hepatic gene expression responsible for b-oxidation of fatty acids in peroxisomes and mitochondria. Enhancement of oxidative processes caused a 2-fold increase in the rate of reactive oxygen species (ROS) production, as evidenced by determination of the level of lipid peroxidation (LPO) products in the liver. Mitochondrial antioxidant systems are more sensitive to elevated ROS production, as they respond by increased expression of SOD2 and PRDX3 genes, than cytoplasmic and peroxisomal antioxidant systems, where expression of CAT1, SOD1, PRDX5 genes remained unaltered.

Abstract 4460: Targeting the redox-protective protein MTH1 for cancer therapy: A novel way to exploit the unique redox status of cancer cells

Abstract Cancer cells are characterized by an increase in the rate of reactive oxygen species (ROS) production and an altered redox environment compared to normal cells. The role of ROS in tumorigenesis is two-fold. On the one hand, ROS play a causal role in tumor development and progression by inducing genomic instability and aberrant, pro-tumorigenic signalling. On the other hand, high levels of ROS can also be toxic to cancer cells, oxidizing and damaging both DNA and free nucleotides (dNTPs), which can lead to cell death. MutT Homolog 1 (MTH1) is a redox-protective phosphatase that converts ROS-oxidized nucleotides (8-oxo-dGTP) into their corresponding monophosphates, thus preventing their incorporation into genomic DNA and the resultant DNA damage and cell death. Recent studies have shown that MTH1 is up-regulated in various cancers and its activity is required for cancer cell survival and proliferation. Genetic knockdown of MTH1 and MTH1 small molecule inhibition led to DNA damage and cancer-specific cell death in vitro and in vivo, while having little effect on normal cells with a lower rate of ROS production. Hence, the unique redox status of cancer cells makes them reliant on redox-protective proteins like MTH1, which opens a novel therapeutic window to selectively induce cancer cell death via oxidative stress, while sparing normal cells. In order to validate MTH1 as a potential cancer therapeutic target, a wide range of cancer cells (lung, bone, colon, skin, and breast) were treated with a selective MTH1 inhibitor (TH588) or MTH1 shRNAs, and analysed for proliferation, cell survival, ROS levels and DNA damage. We found that MTH1 inhibition impaired cancer cell proliferation as well as anchorage-independent growth. Furthermore, TH588 induced cancer cell damage, as assessed by DNA damage markers and cancer cell death. Various normal, non-transformed cells (normal bronchial epithelial cells, normal lung fibroblasts and normal human epidermal melanocytes) were not affected by MTH1 inhibition. Antioxidants, which eliminate ROS, reversed the effects on cancer cells, indicating that ROS is the driver of cancer cell death and growth impairment upon MTH1 inhibition. Consequently, combination approaches using the MTH1 inhibitor together with pro-oxidants, further increased cancer cell susceptibility to oxidative stress. Based on our data, a MTH1 inhibitor may have clinical potential as a single agent or in combination with ROS inducers for the treatment of patients with various forms of cancer. Citation Format: Andrea Glasauer, Horst Irlbacher, Anja Richter, Luisella Toschi, Michael Steckel, Andrea Haegebarth. Targeting the redox-protective protein MTH1 for cancer therapy: A novel way to exploit the unique redox status of cancer cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4460. doi:10.1158/1538-7445.AM2015-4460

PKCα promotes generation of reactive oxygen species via DUOX2 in hepatocellular carcinoma

Hepatocellular carcinoma (HCC) remains the second leading cause of cancer-related death worldwide, and elevated rates of reactive oxygen species (ROS) have long been considered as a hallmark of almost all types of cancer including HCC. Protein kinase C alpha (PKCα), a serine/threonine kinase among conventional PKC family, is recognized as a major player in signal transduction and tumor progression. Overexpression of PKCα is commonly observed in human HCC and associated with its poor prognosis. However, how PKCα is involved in hepatocellular carcinogenesis remains not fully understood. In this study, we found that among the members of conventional PKC family, PKCα, but not PKCβI or βII, promoted ROS production in HCC cells. PKCα stimulated generation of ROS by up-regulating DUOX2 at post-transcriptional level. Depletion of DUOX2 abrogated PKCα-induced activation of AKT/MAPK pathways as well as cell proliferation, migration and invasion in HCC cells. Moreover, the expression of DUOX2 and PKCα was well positively correlated in both HCC cell lines and patient samples. Collectively, our findings demonstrate that PKCα plays a critical role in HCC development by inducing DUOX2 expression and ROS generation, and propose a strategy to target PKCα/DUOX2 as a potential adjuvant therapy for HCC treatment.

Acetylation at lysine 71 inactivates superoxide dismutase 1 and sensitizes cancer cells to genotoxic agents.

Cancer cells are characterized by a high dependency on antioxidant enzymes to cope with the elevated rates of reactive oxygen species (ROS). Impairing antioxidant capacity in cancer cells disturbs the ROS homeostasis and exposes cancer cells to massive oxidative stress. In this study, we have discovered that superoxide dismutase 1 (SOD1), a major player in maintaining the cellular redox status, was acetylated at lysine 71. This acetylation, which was primarily deacetylated by Sirtuin 1 (SIRT1), suppressed the enzymatic activity of SOD1 via disrupting its association with copper chaperone for SOD1 (CCS). More importantly, genotoxic agents, such as camptothecin (CPT), induced SOD1 acetylation by disrupting its binding with SIRT1. CPT-induced SOD1 acetylation was stimulated by its provoked ROS, suggesting a positive feedback loop, in which ROS per se impairs the antioxidative defence of cancer cells and reinforces oxidative stress stimulated by anticancer agents. The intrinsic abundance of SOD1 acetylation varied among cancer cells, and high level of SOD1 acetylation was correlated with elevated sensitivity to CPT. Together, our findings gained mechanistic insights into how cytotoxic agents fine tune the intracellular ROS homeostasis to strengthen their anticancer effects, and suggested SOD1 acetylation as a candidate biomarker for predicting response to CPT-based chemotherapy.

Mechanisms and costs of mitochondrial thermal acclimation in a eurythermal killifish (Fundulus heteroclitus)

Processes acting at the level of the mitochondria have been suggested to affect the thermal limits of organisms. To determine whether changes in mitochondrial properties could underlie shifts in thermal limits, we examined how mitochondrial properties are affected by thermal acclimation in the eurythermal killifish, Fundulus heteroclitus - a species with substantial plasticity in whole-organism thermal limits. We hypothesized that thermal acclimation would result in functional changes in the mitochondria that could result in trade-offs in function during acute thermal shifts. We measured the mitochondrial respiration rate (V̇O2 ) through multiple complexes of the electron transport system following thermal acclimation (to 5, 15, 33°C) and assessed maintenance of mitochondrial membrane potential (Δp) and rates of reactive oxygen species (ROS) production as an estimate of costs. Acclimation to 5°C resulted in a modest compensation of mitochondrial respiration at low temperatures, but these mitochondria were able to maintain Δp with acute exposure to high temperatures, and ROS production did not differ between acclimation groups, suggesting that these increases in mitochondrial capacity do not alter mitochondrial thermal sensitivity. Acclimation to 33°C suppressed mitochondrial respiration as a result of effects on NADH dehydrogenase (complex I). These high-temperature acclimated fish nonetheless maintained levels of Δp and ROS production similar to those of the other acclimation groups. This work demonstrates that killifish mitochondria can successfully acclimate to a wide range of temperatures without incurring major functional trade-offs during acute thermal shifts and that high-temperature acclimation results in a suppression of metabolism, consistent with patterns observed at the organismal level.

Higher Mitochondrial Membrane Potential Induces ROS Production in the Familiar Form of Frontotemporal Dementia with MAPT Mutations

Frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17) is a neurodegenerative disorder caused by mutations in the MAPT gene encoding tau protein. Mitochondrial alterations have been associated with neuronal death in several diseases. The objective of our study was to analyse the mitochondrial function in human iPS cells from a patient of FTDP-17 carrying the 10+16 MAPT mutation. In addition, we have selected three different time points of the differentiation from pluripotent stem cells to cortical neurons to study how mitochondrial alterations develop.We have used fluorescence imaging techniques to examine the mitochondrial function: TMRM to measure the mitochondrial membrane potential (Δψm) and dyhidroethidium (DHE) to measure the rate of reactive oxygen species (ROS) production.Δψm was higher in iPS-derived neurons from the patient bearing the MAPT mutation (158.3±30.2% of control). Higher Δψm was also found in non-differentiated pluripotent stem cells (133.4±10.1%) and in the neural rosettes, which represent an earlier stage of the differentiation (151.5±12.4%). In contrast, mitochondrial mass was lower in mutant iPS-derived neurons (85.1±3.9%), although it was similar in non-differentiated cells.We have also found that the rate of ROS production, measured using DHE, was also higher in iPS-derived neurons from the patient (127±13.9% of control). The increased rate of ROS production in these cells may be the consequence of the enhanced membrane potential. Consistently, the rate of ROS production in non-differentiated cells and in neural rosettes was also significantly higher (123±12.9% and 130±6.9%, respectively).Our study indicates that this MAPT mutation leads to a higher mitochondrial membrane potential, which induces a higher ROS production that may be a trigger for neurodegeneration.

<italic>In situ</italic> microcalorimetric investigation on the photocatalysis with efficient visible-light-driven Ag@AgCl

Visible-light-driven Ag@AgCl deposited Ag NPs on cubic AgCl was prepared via the reduction of precursor AgCl by glucose at room temperature, and it was characterized by X-ray diffraction (XRD) and field-emission scanning electron microscopy (FE-SEM). <italic>In situ</italic> characteristic thermal spectrum and accurate thermokinetic information of three luminous power regarding the photocatalysis of methyl orange were obtained by first employing a novel photoreaction-microcalorimetric system designed by our laboratory. Associated with dynamic information acquired by UV spectrum, and rationally combined the fore three mentioned with the feature of step-wise photodegradation of methyl orange, the photocatalytic mechanism was discussed. The results showed that the initial destruction of azo double band via rapidly endothermic reaction under visible light and followed by a exothermic stage of the oxidation of intermediate products which finally kept a constant exothermic rate for at least 5 h. The procedure of this photocatalysis was influenced by the comprehensive factors of luminous power, mass transfer and diffusion, and the generation rate of reactive oxygen species. The variation of heat effect were lagged as the decrease of luminous power, simultaneously the photodegradation rate slowed down, which took much longer to reach the maximum endothermic peak, exothermic peak, and completely degrade methyl orange.

Correlation between reactive oxygen species production and photochemistry of photosystems I and II in Lemna gibba L. plants under salt stress

Increased rate of reactive oxygen species (ROS) is a common plant response to various environmental stresses. In chloroplast thylakoids, the reaction centers of photosystems I and II are the major generation site of ROS. In the present study, the changes of chlorophyll a fluorescence parameters, P700 absorption change and ROS production (using the fluorescent probe 2,7-dichlorofluorescin diacetate) were investigated in Lemna gibba plants exposed to salt stress (0–400mM NaCl). Salt stress inhibited PSI and PSII activities and resulted in a decrease in overall activity of the electron transport chain while stimulating ROS production. When L. gibba plants were kept into dark condition, NaCl treatment did not showed any significant change in ROS formation compared to control. However, NaCl treatment in light condition induced a strong increase in ROS formation. The production of ROS at 400mM NaCl was 2.6 and 10 fold higher compared to the control respectively after 6h and 24h treatment in light. Furthermore, the correlation between ROS production and the two photosystems (PSI and PSII) activities in L. gibba plants was analyzed. Our data confirmed the correlation between the ROS production and PSII and PSI activities. We showed that ROS production was highly correlated to maximal quantum yield of PSII (R2=0.91) and efficiency with which a trapped exciton can move an electron into the electron transport chain (R2=0.86). While the correlation coefficient (R2) value ROS formation and ΔI/Io820nm (measure of redox states of plastocyanin and P700) was 0.63.

Sirt3 controls chromosome alignment by regulating spindle dynamics during mitosis

Sirt3, one of mammalian sirtuins is a prominent mitochondrial deacetylase that controls mitochondrial oxidative pathways and the rate of reactive oxygen species. Sirt3 also regulates energy metabolism by deacetylating enzymes involved in the metabolic pathway related with lifespan. We report here a novel function of Sirt3 which was found to be involved in mitosis. Depletion of the Sirt3 protein generated unaligned chromosomes in metaphase which caused mitotic arrest by activating spindle assembly checkpoint (SAC). Furthermore, the shape and the amount of the spindles in Sirt3 depleted cells were abnormal. Microtubule (MT) polymerization also increased in Sirt3 depleted cells, suggesting that Sirt3 is involved in spindle dynamics. However, the level of acetylated tubulin was not increased significantly in Sirt3 depleted cells. The findings collectively suggest that Sirt3 is not a tubulin deacetylase but regulates the attachment of spindle MTs to the kinetochore and the subsequent chromosome alignment by increasing spindle dynamics.

Chapter One - The Mitochondrial Free Radical Theory of Aging

The mitochondrial free radical theory of aging is reviewed. Only two parameters currently correlate with species longevity in the right sense: the mitochondrial rate of reactive oxygen species (mitROS) production and the degree of fatty acid unsaturation of tissue membranes. Both are low in long-lived animals. In addition, the best-known manipulation that extends longevity, dietary restriction, also decreases the rate of mitROS production and oxidative damage to mtDNA. The same occurs during protein restriction as well as during methionine restriction. These two manipulations also increase maximum longevity in rodents. The decrease in mitROS generation and oxidative stress that takes place in caloric restriction seems to be due to restriction of a single dietary substance: methionine. The information available supports a mitochondrial free radical theory of aging focused on low generation of endogenous damage and low sensitivity of membranes to oxidation in long-lived animals.

Protective effects of phenolic constituents from Cytisus multiflorus, Lamium album L. and Thymus citriodorus on liver cells

The present study investigated the antioxidant and cytoprotective effects of purified ethanolic extracts of Cytisus multiflorus, Lamium album L. and Thymus citriodorus plants. These extracts showed high antioxidant activity in DPPH and reducing power assays. Using a model of chemical stress induced by potassium dichromate (DK) in human hepatoblastoma HepG2 cells, 50μg/mL of C. multiflorus, L. album and T. citriodorus extracts decreased the rate of reactive oxygen species (ROS) production by 35%, 26% and 20%, respectively, when exposed to 25μM of DK. This effect was also observed for the treatment of cells with individual polyphenolic compounds determined in the extracts, or with mixtures prepared with individual polyphenolic compounds simulating the phenolic composition of the extracts. Additionally, the purified ethanolic extracts and the prepared polyphenolic mixtures showed a cytoprotective effect against DK-induced toxicity. The overall results emphasize the contribution of polyphenols in antioxidant and cytoprotective properties of the studied plants.

Guanosine diphosphate exerts a lower effect on superoxide release from mitochondrial matrix in the brains of uncoupling protein-2 knockout mice: New evidence for a putative novel function of uncoupling proteins as superoxide anion transporters

In this report, we show new experimental evidence that, in mouse brain mitochondria, uncoupling protein-2 (UCP2) can be involved in superoxide (O2-) removal from the mitochondrial matrix. We found that the effect of guanosine 5′-diphosphate (GDP) on the rate of reactive oxygen species (ROS) release from brain mitochondria of UCP2 knockout mice was less pronounced compared to the wild type animals. This putative novel UCP2 activity, evaluated by the use of UCP2-knockout transgenic animals, along with the known antioxidant defence systems, may provide additional protection from ROS in brain mitochondria.

Association of Mitochondrial DNA Variations with Lung Cancer Risk in a Han Chinese Population from Southwestern China

Mitochondrial DNA (mtDNA) is particularly susceptible to oxidative damage and mutation due to the high rate of reactive oxygen species (ROS) production and limited DNA-repair capacity in mitochondrial. Previous studies demonstrated that the increased mtDNA copy number for compensation for damage, which was associated with cigarette smoking, has been found to be associated with lung cancer risk among heavy smokers. Given that the common and “non-pathological” mtDNA variations determine differences in oxidative phosphorylation performance and ROS production, an important determinant of lung cancer risk, we hypothesize that the mtDNA variations may play roles in lung cancer risk. To test this hypothesis, we conducted a case-control study to compare the frequencies of mtDNA haplogroups and an 822 bp mtDNA deletion between 422 lung cancer patients and 504 controls. Multivariate logistic regression analysis revealed that haplogroups D and F were related to individual lung cancer resistance (OR = 0.465, 95%CI = 0.329–0.656, p<0.001; and OR = 0.622, 95%CI = 0.425–0.909, p = 0.014, respectively), while haplogroups G and M7 might be risk factors for lung cancer (OR = 3.924, 95%CI = 1.757–6.689, p<0.001; and OR = 2.037, 95%CI = 1.253–3.312, p = 0.004, respectively). Additionally, multivariate logistic regression analysis revealed that cigarette smoking was a risk factor for the 822 bp mtDNA deletion. Furthermore, the increased frequencies of the mtDNA deletion in male cigarette smoking subjects of combined cases and controls with haplogroup D indicated that the haplogroup D might be susceptible to DNA damage from external ROS caused by heavy cigarette smoking.

Comparative study on metal homeostasis and detoxification in two Antarctic teleosts

The main characteristic of Antarctic seawater is its low constant temperature and its high concentration of O2, which can increase the formation rate of reactive oxygen species (ROS), together with a natural occurrence of elevated cadmium and copper levels. In the present paper, we studied the presence of cadmium, copper and zinc, metallothioneins (MTs) and glutathione (GSH), and antioxidant enzyme activities in the Antarctic teleosts Trematomus bernacchii and Trematomus newnesi, in order to determine the influence of the peculiar physico-chemical features of the Antarctic marine environment on these physiological defence systems in two species of teleosts. In both of them, cadmium and copper accumulation occurs mostly in the liver. T. bernacchii accumulates zinc mostly in the hepatic tissue, whereas T. newnesi does not show a preferential accumulation site. In addition to the intra-specific analysis, we decided to compare the two species of the Trematomus genus in order to verify if the different feeding habits and motility of these fish affects metal accumulation. Our results show that the liver of T. bernacchii accumulates cadmium and zinc at a higher extent with respect to T. newnesi. Glutathione (GSH) and metallothioneins (MTs) are present in great quantity in the liver of both species. Moreover liver is the tissue which generally showed the highest antioxidant enzyme levels. The results provide further insights in the physiological mechanisms evolved by animals living in this extreme environment.

Iontophoretic transport of zinc phthalocyanine tetrasulfonic acid as a tool to improve drug topical delivery

Phthalocyanines have been used as systemic photosensitizers because of their high affinity towards tumour tissue, and the high rates of reactive oxygen species produced when they are irradiated during photodynamic therapy. However, the topical administration of these compounds is limited by their large size, poor hydrosolubility and ionic character. This study aimed to investigate the iontophoretic delivery of charged zinc phthalocyanine tetrasulfonic acid (ZnPcS4) from a hydrophilic gel to different skin layers by means of in-vitro and in-vivo studies. Six hours of passive administration was insufficient for ZnPcS4 to cross the stratum corneum (SC) and to reach the epidermis and dermis. No positive effect was reached when anodal iontophoresis was performed, showing that the drug-electrode attraction effect was higher than the electro-osmosis contribution at a pH of 5.5. Cathodal iontophoresis, however, was able to transport significant amounts of the drug to the viable epidermis. In addition, the absence of NaCl in the formulation significantly increased (by five-fold) the amount of ZnPcS4 that crossed the SC and accumulated in the epidermis and dermis. It was possible to visualize the drug accumulation in the follicle openings and in the epidermis, even after SC removal. In-vivo experiments in rat skin showed that these results were maintained in an in-vivo model, even with only 15 min of iontophoresis. In addition, confocal analysis of the treated skin showed a homogeneous distribution of ZnPcS4 in the viable epidermis after this short period of cathodal iontophoresis.

An evolutionary comparative scan for longevity-related oxidative stress resistance mechanisms in homeotherms

Key mechanisms relating oxidative stress to longevity from an interespecies comparative approach are reviewed. Long-lived animal species show low rates of reactive oxygen species (ROS) generation and oxidative damage at their mitochondria. Comparative physiology also shows that the specific compositional pattern of tissue macromolecules (proteins, lipids and nucleic acids) in long-lived animal species gives them an intrinsically high resistance to modification that likely contributes to their superior longevity. This is obtained in the case of lipids by decreasing the degree of fatty acid unsaturation, and in the case of proteins by lowering their methionine content. These findings are also substantiated from a phylogenomic approach. Nutritional or/and pharmacological interventions focused to modify some of these molecular traits were translated with modifications in animal longevity. It is proposed that natural selection tends to decrease the mitochondrial ROS generation and to increase the molecular resistance to the oxidative damage in long-lived species.

Multiphoton Imaging of the Functioning Kidney

Translating discoveries made in isolated renal cells and tubules to the in vivo situation requires the assessment of cellular function in intact live organs. Multiphoton imaging is a form of fluorescence microscopy that is ideally suited to working with whole tissues and organs, but adequately loading cells with fluorescence dyes in vivo remains a challenge. We found that recirculation of fluorescence dyes in the rat isolated perfused kidney (IPK) resulted in levels of intracellular loading that would be difficult to achieve in vivo. This technique allowed the imaging of tubular cell structure and function with multiphoton microscopy in an intact, functioning organ. We used this approach to follow processes in real time, including (1) relative rates of reactive oxygen species (ROS) production in different tubule types, (2) filtration and tubular uptake of low-molecular-weight dextrans and proteins, and (3) the effects of ischemia-reperfusion injury on mitochondrial function and cell structure. This study demonstrates that multiphoton microscopy of the isolated perfused kidney is a powerful technique for detailed imaging of cell structure and function in an intact organ.

Reactive Oxygen Species Is Essential for Cycloheximide to Sensitize Lexatumumab-Induced Apoptosis in Hepatocellular Carcinoma Cells

This study aims to investigate apoptosis induced by lexatumumab (Lexa) in hepatocellular carcinoma (HCC) cells. We assessed the sensitivity of HCC cell lines and normal human hepatocytes to Lexa and explored the sensitization of HCC cells to Lexa-induced apoptosis by cycloheximide (CHX). Our data indicated that CHX sensitized HCC cell lines to Lexa-induced apoptosis, whereas treatment using solely CHX or Lexa was ineffective. The sequential treatment of CHX followed by Lexa dramatically induced caspase-dependent apoptosis in HCC cells and had synergistically increased intracellular rates of reactive oxygen species (ROS). Additionally, when ROS production was blocked by N-acetyl-L-cysteine (NAC), HCC cells were protected against Lexa and CHX combination treatment-induced apoptosis. ROS generation induced by combination treatment of Lexa and CHX triggered pro-apoptotic protein Bax oligomerization, conformation change, and translocation to mitochondria, which resulted in the release of cytochrome c and subsequent cell death. Furthermore, HSP90 was involved in mediating Lexa and CHX combination treatment-induced ROS increase and apoptotic death. More importantly, we observed that combination treatment of Lexa and CHX did not cause apoptotic toxicity in normal human primary hepatocytes. These results suggest that Lexa and CHX combination treatment merits investigation for the development of therapies for patients with HCC.

Vascular reactivity and metabolism of the reactive forms of oxygen and nitrogen: effects of low doses of radiation

Endothelium-dependent and endothelium-independent vascular reactions of relaxation, the rate of reactive oxygen species (superoxide and hydroxyl) generation, the content of stable nitric oxide (NO) metabolites (nitrite, nitrate and nitrosothiols), the pools of lipid peroxidation products (eicosanoids LTC 4 and TxB 2 , dienic conjugates and malonic dialdehyde), H 2 O 2 , uric acid and urea were studied in aorta preparations of BALB/c 9 months-old mice: group I – control; group II – exposed to chronic external low doses γ -irradiation (equivalent dose of 96.9 μSv·h –1 ) mice (cumulative dose of 0.43 Sv). The results obtained showed disturbances in the studied reactions and change of the reactive forms of oxygen and nitrogen following an effect of low doses of radiation. The latter induce significant changes in the pools of NO stable metabolites, which can cause disturbances in the NO-dependent physiological functions of heart and aorta. A significant decrease in the levels of nitrite and S-nitrosothiols can result in nitrosative stress.