Oxidative Stress in the Developing Rat Brain due to Production of Reactive Oxygen and Nitrogen Species.

Oxidative stress after birth led us to localize reactive oxygen and nitrogen species (RONS) production in the developing rat brain. Brains were assessed a day prenatally and on postnatal days 1, 2, 4, 8, 14, 30, and 60. Oxidation of dihydroethidium detected superoxide; 6-carboxy-2',7'-dichlorodihydrofluorescein diacetate revealed hydrogen peroxide; immunohistochemical proof of nitrotyrosine and carboxyethyllysine detected peroxynitrite formation and lipid peroxidation, respectively. Blue autofluorescence detected protein oxidation. The foetuses showed moderate RONS production, which changed cyclically during further development. The periods and sites of peak production of individual RONS differed, suggesting independent generation. On day 1, neuronal/glial RONS production decreased indicating that increased oxygen concentration after birth did not cause oxidative stress. Dramatic changes in the amount and the sites of RONS production occurred on day 4. Nitrotyrosine detection reached its maximum. Day 14 represented other vast alterations in RONS generation. Superoxide production in arachnoidal membrane reached its peak. From this day on, the internal elastic laminae of blood vessels revealed the blue autofluorescence. The adult animals produced moderate levels of superoxide; all other markers reached their minimum. There was a strong correlation between detection of nitrotyrosine and carboxyethyllysine probably caused by lipid peroxidation initiated with RONS.

Lipophilic fluorescent products of free radicals.

BACKGROUND: Fluorescent pigments are the end-products of reactions involving free radical attack on biological molecules and can be formed, for example, in reactions between lipid peroxidation products, mainly unsaturated aldehydes, with free amino groups. Their characteristic emission maximum was found to be at 420-470 nm after being excited at 340-390 nm. The mechanism of their formation and chemical identity has been revealed in many in vitro studies, in which reactive aldehydes were incubated with amino group-containing molecules. Owing to their intrinsic fluorescent properties and molecular stability these products are easily measured by means of spectrofluorimetry and are used as biomarkers of oxidative stress caused by various triggers. It has been found that the fluorescent products are formed in excess in conditions linked with increased free radical production, such as atherosclerosis, Alzheimer's disease and multiple sclerosis. METHODS: We searched the literature using "MEDLINE" and "Web of Science" in order to get an overview of the state of knowledge about fluorescent products of free radicals, that is, their analysis from in vitro studies, animal and human studies and their use as markers of oxidative damage. CONCLUSIONS: Although their chemical structure may not have been elucidated, the fluorophores formed in this way have found application as markers of oxidative stress in many animal and human studies. In vitro experiments using model reactions have given some clues as to how certain fluorescent pigments arise during oxidative reactions in vivo. Advances in analytical techniques should lead the chemical characterization of pigments of different origin to completeness.

Evaluation of lipofuscin-like pigments as an index of lead-induced oxidative damage in the brain.

This study was carried out to investigate the role of lead in the development of oxidative stress in the brain. We examined the rate of lipid peroxidation and we determined lipid fluorescence products (lipofuscin-like pigments - LFP) as a marker of lipid peroxidation after short in vitro incubation of rat brain homogenates with lead acetate (10(-2), 10(-4), 10(-6) M lead acetate, 2 h). Simultaneously we examined by the same method in vivo indices of oxidative stress in brains of mice exposed for 12 weeks to 0.2% lead acetate in drinking water. The results show that the concentration of LFP in rat brain homogenates increased significantly after 2 h incubation with 10(-2) M lead acetate as compared to controls (P<0.0001). This effect was not observed in lower doses of lead acetate (10(-4) and 10(-6) M). After the long-term exposure of mice to 0.2% lead acetate, pronounced accumulation of lead and significantly increased concentration of LFP (P<0.004) in the brains of exposed animals as compared to controls were observed. The evidence for the formation of specific fluorophores originating from oxidative damage was shown also in qualitative changes in 3D spectral arrays and synchronous spectra. The presented results proved the influence of lead on the activation of radical reactions in the brain after short in vitro exposure of rat brain as well as within long-term in vivo exposure in mice using lipofuscin-like pigments as an indicator of oxidative stress.

Generation of hydrogen peroxide in the developing rat heart: the role of elastin metabolism.

Reports describing production of reactive oxygen species in neonatal heart are missing. As lysyl oxidase is potentially important source of H(2)O(2), we studied its role during ontogenic development of rat heart. H(2)O(2) was detected in thin sections of developing rat heart by fluorescence microscopy with the use of fluorescence probe 2'-7'-dichlorofluorescin. The experimental design comprised foetuses 21 days after conception, and then the animals sampled on the 1st, 4th, 7th, 10th, 15th, 30th and 60th day after birth. We also used 7-month-old animals as an example of ageing effects. Since the day 4 on, H(2)O(2) was produced only extracellularly up to the day 15, between days 30 and 60 intracellular production was detected as well, and in 7-month-old animals only extracellular production was observed. The specific inhibitors of lysyl oxidase almost completely quenched the H(2)O(2)-dependent fluorescence. Starting from day 7, blue autofluorescence specific to oxidized proteins developed in the vessel wall. Intracellular blue autofluorescence specific to autoxidation products developed after day 30. Chloroform extraction diminished the intracellular blue fluorescence, leaving the extracellular fluorescence intact. This confirmed the protein nature of the fluorophores. Lysyl oxidase is significant source of H(2)O(2) in the heart vessel wall during development and H(2)O(2) oxidatively modifies elastin producing protein blue autofluorescence.

Early postnatal development of rat brain is accompanied by generation of lipofuscin-like pigments.

The increased generation of free radicals results in the formation of fluorescent end-products of lipid peroxidation, lipofuscin-like pigments (LFPs). The authors observed that LFPs are generated in rat brain after a normal birth during 5 postnatal days. The experimental design of the study comprised 10 groups of animals. The authors measured prenatal values 1 day and 7 days before birth, and then the animals were sampled on postnatal day 1, 2, 5, 10, 15, 25, 35, and 90. Maximum LFP concentration is achieved on the postnatal day 2. Starting from postnatal day 10, LFP concentration returns to prenatal values. A new rise in LFP concentration is observed at 3 months of age. This is associated with the beginning of the aging process. LFPs were characterized by fluorescence spectroscopy using tridimensional excitation spectra, synchronous spectra and their derivatives, and HPLC with fluorescence detection. It was possible to discern several tens of fluorescent compounds of unknown structure that are generated and metabolized during early development. The authors suggest that LFPs are formed after respiratory burst of microglia phagocytosing apoptotic cells.

Evaluation of different methods detecting intracellular generation of free radicals.

Reactive oxygen species (ROS) play several biological roles. We investigated the applicability of fluorescent probes for their detection (i) in rabbit lens epithelial cells during ageing in culture, and (ii) in thin sections of rat heart. We used dihydroethidium (DHE), dichlorofluorescin (DCFH), and dihydrorhodamine 123 (DHR) together with detection of autofluorescence both in cells and in chloroform extracts. Superoxide production was confirmed by a specific histochemical method using Mn(2+). All methods demonstrated higher production of ROS in older cells. All probes revealed different sites of ROS production in young and old cells and could be used for investigation of ROS generation during cell ageing. In the thin sections of rat heart DCFH was not suitable for intracellular ROS detection. The results indicate that the potential of fluorescent dyes in ROS detection is not usually fully exploited, and that blue autofluorescence is associated with oxidative damage.

Hypercapnia protects erythrocytes against free radical damage induced by hypoxia in exposed rats.

Several studies report that hypoxic exposure induces free radical oxidative damage in various tissues. The mechanism of this damage includes membrane lipid peroxidation which can be easily detected by measuring fluorescent end-products of the process, i.e. lipofuscin-like pigments. Four day exposure of rats to hypoxia (10% O(2)) increased the level of lipofuscin-like pigments in erythrocytes up to 9 fold. This increase was completely prevented when the animals were exposed to hypercapnia (4.3% CO(2)) in addition to hypoxia. We studied the possible mechanism of the hypercapnic protection on isolated erythrocyte membranes in vitro. Lipid peroxidation was initiated by incubation of the membranes with iron ions and ascorbate. Production of malonaldehyde, the precursor of lipofuscin-like pigments, was strongly inhibited in bicarbonate buffer. Similarly the production of lipofuscin-like products was damped. These experiments suggest that the protective effect of hypercapnia might consist in direct interaction of CO(2) with free radical processes.

Role of oxidative stress in PKC-delta upregulation and cardioprotection induced by chronic intermittent hypoxia.

The aim was to determine whether increased oxidative stress during the adaptation to chronic intermittent hypoxia (CIH) plays a role in the induction of improved cardiac ischemic tolerance. Adult male Wistar rats were exposed to CIH in a hypobaric chamber (7,000 m, 8 h/day, 5 days/wk, 24-30 exposures). Half of the animals received antioxidant N-acetylcysteine (NAC; 100 mg/kg) daily before the exposure; the remaining rats received saline. Control rats were kept under normoxia and treated in a corresponding manner. One day after the last exposure (and/or NAC injection), anesthetized animals were subject to 20 min of coronary artery occlusion and 3 h of reperfusion for determination of infarct size. In parallel subgroups, biochemical analyses of the left ventricular myocardium were performed. Adaptation to CIH reduced infarct size from 56.7 +/- 4.5% of the area at risk in the normoxic controls to 27.7 +/- 4.9%. NAC treatment decreased the infarct size in the controls to 42.0 +/- 3.4%, but it abolished the protection provided by CIH (to 41.1 +/- 4.9%). CIH decreased the reduced-to-oxidized glutathione ratio and increased the relative amount of PKC isoform-delta in the particulate fraction; NAC prevented these effects. The expression of PKC-epsilon was decreased by CIH and not affected by NAC. Activities of superoxide dismutase, catalase, and glutathione peroxidase were affected by neither CIH nor NAC treatment. It is concluded that oxidative stress associated with CIH plays a role in the development of increased cardiac ischemic tolerance. The infarct size-limiting mechanism of CIH seems to involve the PKC-delta-dependent pathway but apparently not the increased capacity of major antioxidant enzymes.

Selenium protects the immature rat heart against ischemia/reperfusion injury.

The aim of the study was to find out whether administration of selenium (Se) will protect the immature heart against ischemia/reperfusion.The control pregnant rats were fed laboratory diet (0.237 mg Se/kg diet); experimental rats received 2 ppm Na(2)SeO(3) in the drinking water from the first day of pregnancy until day 10 post partum. The concentration of Se in the serum and heart tissue was determined by activation analysis, the serum concentration of NO by chemiluminescence, cardiac concentration of lipofuscin-like pigment by fluorescence analysis. The 10 day-old hearts were perfused (Langendorff); recovery of developed force (DF) was measured after 40 min of global ischemia. In acute experiments, 10 day-old hearts were perfused with selenium (75 nmol/l) before or after global ischemia. Sensitivity to isoproterenol (ISO, pD(50)) was assessed as a response of DF to increasing cumulative dose.Se supplementation elevated serum concentration of Se by 16%. Se increased ischemic tolerance (recovery of DF, 32.28 +/- 2.37 vs. 41.82 +/- 2.91%, P < 0.05). Similar results were obtained after acute administration of Se during post-ischemic reperfusion (32.28 +/- 2.37 vs. 49.73 +/- 4.40%, P < 0.01). The pre-ischemic treatment, however, attenuated the recovery (23.08 +/- 3.04 vs. 32.28 +/- 2.37%, P < 0.05). Moreover, Se supplementation increased the sensitivity to the inotropic effect of ISO, decreased cardiac concentration of lipofuscin-like pigment and serum concentration of NO. Our results suggest that Se protects the immature heart against ischemia/reperfusion injury. It seems therefore, that ROS may affect the function of the neonatal heart, similarly as in adults.

The effects of reactive oxygen and nitrogen species during yeast replicative ageing.

Free radicals are considered the most important cause of cellular ageing. We have investigated ageing process in the yeast Saccharomyces cerevisiae. We have compared the wild type strain with the mutant cells with constitutively active Ras oncogen, which generates increased amounts of free radicals. Increased generation of oxygen-derived free radicals resulted in the Ras mutant cells accumulation of lipofuscin-like pigments during ageing. Ageing wild type cells did not accumulate lipofuscin-like pigments. This is quite unique feature among known biological models. It may be caused by increased concentration of alpha tocopherol (the most prominent lipophilic antioxidant) in the wild type cells. In contrast, the Ras mutant cells contained decreased levels of alpha tocopherol even in the young cells. This observation indicates that the increased free radical generation can overwhelm the endogenous antioxidant system. We have documented the involvement of nitrogen-derived free radicals in the yeast metabolism. Protein nitrotyrosine, a marker of the reactive nitrogen species, has significantly increased in the senescent Ras mutant cells. The wild type cells contained basic level of nitrotyrosine corresponding to its concentration found in non-activated mammalian macrophages.

Glycerophosphate-dependent hydrogen peroxide production by brown adipose tissue mitochondria and its activation by ferricyanide.

Oxidation of glycerophosphate (GP) by brown adipose tissue mitochondria in the presence of antimycin A was found to be accompanied by significant production of hydrogen peroxide. GP-dependent hydrogen peroxide production could be detected by p-hydroxyphenylacetate fluorescence changes or as an antimycin A-insensitive oxygen consumption. One-electron acceptor, potassium ferricyanide, highly stimulated the rate of GP-dependent antimycin A-insensitive oxygen uptake, which was prevented by inhibitors of mitochondrial GP dehydrogenase (mGPDH) or by coenzyme Q (CoQ). GP-dependent ferricyanide-induced peroxide production was also determined luminometrically, using mitochondria or partially purified mGPDH. Ferricyanide-induced peroxide production was negligible, when succinate or NADH was used as a substrate. These results indicate that hydrogen peroxide is produced directly by mGPDH and reflect the differences in the transport of reducing equivalents from mGPDH and succinate dehydrogenase to the CoQ pool. The data suggest that more intensive production of reactive oxygen species may be present in mammalian cells with active mGPDH.

Ultraviolet light-irradiated collagen III modulates expression of cytoskeletal and surface adhesion molecules in rat aortic smooth muscle cells in vitro.

Systemic and pulmonary hypertension is characterised by structural reconstruction of the vascular wall which includes hypertrophy and hyperplasia of vascular smooth muscle cells (VSMCs) and fibroproduction. We hypothesise that these changes are stimulated by non-enzymatic modification of collagen molecules in the injured vascular wall by radicals. We exposed collagen III to ultraviolet (UV) light irradiation which, as indicated by fluorescence and electrophoretic analyses, resulted in its fragmentation. Both irradiated and control unmodified collagen were adsorbed on culture dishes and seeded with VSMCs derived from the rat thoracic aorta. During the first week after seeding, the cells on the modified collagen attained significantly higher population density (by 15-83%), higher mitotic index (by 31-135%) and higher BrdU labelling index (by 32%). However, these cells were less resistant to spontaneous and trypsin-mediated detachment from the growth support. As revealed using enzyme-linked immunosorbent assay in 3-day-old cultures, the cells growing on the irradiated collagen exhibited a lower concentration of beta-1 integrins (-10%, measured per milligram of protein), vinculin (-18%), talin (-6%) and vimentin (-15%). Immunofluorescence staining showed that these molecules were distributed more diffusely and less organised into focal adhesion plaques or cytoskeletal fibres. The concentration of two adhesion molecules of immunoglobulin type, ICAM-1 and VCAM-1, was increased by 11% and 16%, respectively. The concentration of alpha-v integrins and alpha-actin was unchanged; the latter, however, formed fewer distinct microfilament bundles in cells on the modified collagen. Our results suggest that the VSMCs growing on UV-modified collagen are more prone to escape the growth control mediated by cell-extracellular matrix contact and can bind the cells of the immune system.