Zuzana Tatarkova

Oxidative modifications of cardiac mitochondria and inhibition of cytochrome c oxidase activity by 4-hydroxynonenal

Abstract 4-Hydroxynonenal (HNE) is a highly toxic product of lipid peroxidation (LPO). Its role in the inhibition of cytochrome c oxidase activity and oxidative modifications of mitochondrial lipids and proteins were investigated. The exposure of mitochondria isolated from rat heart to HNE resulted in a time- and concentration-dependent inhibition of cytochrome c oxidase activity with an IC50 value of 8.3 ± 1.0 μM. Immunoprecipitation-Western blot analysis showed the formation of HNE adducts with cytochrome c oxidase subunit I. The loss of cytochrome c oxidase activity was also accompanied by reduced thiol group content and increased HNE-lysine fluorescence. Furthermore, there was a marked increase in conjugated diene formation indicating LPO induction by HNE. Fluorescence measurements revealed the formation of bityrosines and increased surface hydrophobicity of HNE-treated mitochondrial membranes. Superoxide dismutase + catalase and the HO• radical scavenger mannitol partially prevented inhibition of cytochrome c oxidase activity and formation of bityrosines. These findings suggest that HNE induces formation of reactive oxygen species and its damaging effect on mitochondria involves both formation of HNE–protein adducts and oxidation of membrane lipids and proteins by free radicals.

Ischemia-Induced Inhibition of Mitochondrial Complex I in Rat Brain: Effect of Permeabilization Method and Electron Acceptor

In this study we have examined the effect of global brain ischemia/reperfusion on biochemical properties of the mitochondrial respiratory complex I (CI) in rat hippocampus and cortex. Since the inner mitochondrial membrane forms the permeability barrier for NADH, the methodology of enzymatic activity determinations employs membrane permeabilization methods. This action affects the basic character of electrostatic and hydrophobic interactions inside the membrane and might influence functional properties of membrane embedded proteins. Therefore we have performed the comparative analysis of two permeabilization methods (sonication, detergent) and their impact on CI enzymatic activities under global brain ischemic-reperfusion conditions. We have observed that ischemia led to significant decrease of CI activities using both permeabilization methods in both brain areas. However, significant differencies in enzymatic activities were registered during reperfusion intervals according to used permeabilization method. We have also tested the effect of electron acceptors (decylubiquinone, potassium ferricyanide, nitrotetrazolium blue) on CI activities during I/R. Based on our results we assume that the critical site where ischemia affects CI activities is electron transfer to electron acceptor. Further, the observed mitochondrial dysfunction was analyzed by means of one and 2-dimensional BN PAGE/SDS PAGE with the focus on 3-nitrotyrosine immunodetection as a marker of oxidative damage to proteins. Add to this, initialization of p53 mitochondrial apoptosis through p53, Bax, Bcl-XL proteins and a possible involvement of GRIM-19, the CI structural subunit, in apoptotic processes were also studied.

Response of secretory pathways Ca(2+) ATPase gene expression to hyperhomocysteinemia and/or ischemic preconditioning in rat cerebral cortex and hippocampus.

The study determines whether hyperhomocysteinemia (risk factor of brain ischemia) alone or in combination with ischemic preconditioning (IPC) affects the ischemia-induced changes in gene expression of secretory pathways Ca(2+)-ATPase (SPCA1). Hyperhomocysteinemia was induced by subcutaneous administration of homocysteine (Hcy; 0.45 µmol/g body weight) twice a day at 8 h intervals for 14 days. Rats were preconditioned by 5 min ischemia and 2 days later, 15 min of global forebrain ischemia was induced by four vessel occlusion. We observed that hyperhomocysteinemia significantly decreased the level of SPCA1 mRNA in the cortex. Pre-ischemic challenge was noticeable in both brain areas. In the cortex, pre-ischemia in Hcy group led to the abrupt stimulation of the mRNA expression by 249% within the Hcy ischemic group and by 321% in the Hcy control. Values further exceeded those observed in the naive control. In the hippocampus, the differences between naive and Hcy groups were not observed. IPC initiated elevation of mRNA expression to 159% (p < 0.05) of control with Hcy and to 131% (p < 0.01) of ischemia with Hcy, respectively. Documented response of SPCA gene to IPC in hyperhomocysteinemic group might suggest a correlation of SPCA expression consistent with the role of cross-talks between intracellular Ca(2+) stores including secretory pathways in the tolerance phenomenon.

The Effect of Aging on Mitochondrial Complex I and the Extent of Oxidative Stress in the Rat Brain Cortex

One of the characteristic features of the aging is dysfunction of mitochondria. Its role in the regulation of metabolism and apoptosis suggests a possible link between these cellular processes. This study investigates the relationship of respiratory complex I with aging-related oxidative stress in the cerebral mitochondria. Deterioration of complex I seen in senescent (26-months old) mitochondria was accompanied by decline in total thiol group content, increase of HNE and HNE-protein adducts as well as decreased content of complex I subunits, GRIM-19 and NDUFV2. On the other hand, decline of complex I might be related with the mitochondrial apoptosis through increased Bax/Bcl-2 cascade in 15-month old animal brains. Higher amount of Bcl-2, Bcl-xL with the lower content of GRIM-19 could maintain to some extent elevated oxidative stress in mitochondria as seen in the senescent group. In the cortical M1 region increased presence of TUNEL+ cells and more than 20-times higher density of Fluoro-Jade C+ cells in 26-months old was observed, suggesting significant neurodegenerative effect of aging in the neuronal cells. Our study supports a scenario in which the age-related decline of complex I might sensitize neurons to the action of death agonists, such as Bax through lipid and protein oxidative stimuli in mitochondria. Although aging is associated with oxidative stress, these changes did not increase progressively with age, as similar extent of lesions was observed in oxidative stress markers of the both aged groups.

Effects of long-term oxygen treatment on α-ketoglutarate dehydrogenase activity and oxidative modifications in mitochondria of the guinea pig heart

ObjectiveOxygen therapy is used for the treatment of various diseases, but prolonged exposure to high concentrations of O2 is also associated with formation of free radicals and oxidative damage.MethodsIn the present study we compared α-ketoglutarate dehydrogenase (KGDH) activity and mitochondrial oxidative damage in the hearts of guinea pigs after long-term (17 and 60 h) oxygenation with 100% normobaric O2 and with partially negatively (O2 neg) or positively (O2 posit) ionized oxygen.ResultsInhalation of O2 led to significant loss in KGDH activity and thiol group content and accumulation of bityrosines. Inhalation of O2 neg was accompanied by more pronounced KGDH inhibition, possibly due to additional formation of protein-lipid conjugates. In contrast, O2 posit prevented loss in KGDH activity and diminished mitochondrial oxidative damage.ConclusionsThese findings suggest that oxygen treatment is associated with impairment of heart energy metabolism and support the view that inhalation of O2 posit optimizes the beneficial effects of oxygen therapy.

Effect of normobaric oxygen treatment on oxidative stress and enzyme activities in guinea pig heart.

Normobaric oxygen (NBO) therapy is commonly applied for the treatment of various diseases, including myocardial infarctions, but its effectiveness is controversial. Potential adverse effects of hyperoxia are related to excessive formation of free radicals. In the present study we examined the effect of 60-h NBO treatment on lipid peroxidation (LPO), activity of manganese superoxide dismutase (Mn-SOD) and mitochondrial enzymes of energy metabolism in guinea pig heart. NBO treatment resulted in significant accumulation of thiobarbituric acid reactive substances and loss of Mn-SOD activity despite slight elevation of Mn-SOD protein content. Activity of electron transport chain complex III decreased significantly, while activity of complex IV was slightly elevated and citrate synthase was unchanged. LPO, inhibition of Mn-SOD and complex III activities were more pronounced when inhaled oxygen was partially enriched with superoxide radical. In contrast, when O(2) was enriched with oxygen cation (O(2)●+), LPO and loss of Mn-SOD activity were prevented. Complex III activity in the O(2)●+-treated group remained depressed but activities of complex IV and citrate synthase were elevated. These data suggest that NBO treatment is associated with myocardial oxidative damage and attenuation of antioxidant defense, but these adverse effects can be partially attenuated by inhalation of O(2) enriched with oxygen cation.

The Involvement of Mg2

Mg2+ is an essential mineral with pleotropic impacts on cellular physiology and functions. It acts as a cofactor of several important enzymes, as a regulator of ion channels such as voltage-dependent Ca2+ channels and K+ channels and on Ca2+-binding proteins. In general, Mg2+ is considered as the main intracellular antagonist of Ca2+, which is an essential secondary messenger initiating or regulating a great number of cellular functions. This review examines the effects of Mg2+ on mitochondrial functions with a particular focus on energy metabolism, mitochondrial Ca2+ handling, and apoptosis.

The role of CYP17A1 in prostate cancer development: structure, function, mechanism of action, genetic variations and its inhibition

Androgens play an important role during the development of both normal prostate epithelium and prostate cancer and variants of genes involved in androgen metabolism may be related to an increased risk of prostate disease. Cytochrome P450 17α-hydroxylase/17,20-lyase (CYP17A1) is a key regulatory enzyme in the steroidogenic pathway; it catalyses both 17α-hydroxylase and 17,20-lyase activities and is essential for the production of both androgens and glucocorticoids. In this review, we focus on the structure and enzymatic activity of CYP17A1 and the mechanism of modulation of CYP17A1 activities. We discuss the relationship between common genetic variations in CYP17A1 gene and prostate cancer risk and the main effects of these variations on the prediction of susceptibility and clinical outcomes of prostate cancer patients. The mechanism of action, the efficacy and the clinical potential of CYP17A1 inhibitors in prostate cancer are also summarized.

Nanomaterials - a New and Former Public Health Issue. the Case of Slovakia

Nanoparticles exist for a long time as both inorganic and organic parts of nature. Recently, massive expansion of nanotechnologies is evidenced, together with intentional production of new nanoparticles which have not been in contact with living organisms until now. Besides obvious positive aspects, potential threats related to their exposure should be taken into consideration. Unique physical-chemical properties of nanoparticles cause a high bioactivity following their intake (through air, ingestion and skin) and unrestricted spread in exposed organs. Primary effects of nanoparticles on cellular level represent oxidative stress and reactions leading to apoptosis, autophagocytosis and necrosis. Number of studies indicating contribution of nanoparticles to numerous disorders has been recently increasing. However, detailed mechanisms of health effects are not well known. Similarly, there is insufficient information on life cycle of nanoparticles in the environment. Research in this field as well as legislation is behind rapid development and use of nanotechnologies. Considering absence of mandatory exposure limits and other protective measures, nanomaterials represent a potential threat for population health. Recommendations and guidelines of international institutions can contribute to deal with situation, however, passing of effective legislation both on national and European level is urgently needed.

Oxidative Damage to Proteins and Lipids During Ageing

Oxidative Damage to Proteins and Lipids During Ageing The present work is a theoretical study in the field of monitoring oxidative damage to proteins and lipids during ageing. The basic terminology and interactions are discussed as well as the sources of oxidants and their elimination through antioxidant protection. We focus on the effects of oxidative stress on the biomolecules (proteins, lipids and DNA), the role of mitochondria, antioxidants, physical activity and caloric restriction in relation to ageing. Previous research indicates the crucial role of mitochondria in the ageing process by their formation of oxidants, the accumulation of oxidative damage to mtDNA and other biomolecules leading to impairment of mitochondrial function, energy failure, apoptosis and necrosis. Although the role of oxidative stress in the ageing process is evident and well documented, the precise mechanisms of its relationships remain largely unknown. Further research is needed to clarify them and to show ways to slow down the ageing process.