Direct measurement of free radical levels in the brain after cortical ischemia induced by photothrombosis.

Tissue ischemia is connected with the production of free radicals (FR). This study was designed to directly measure of the amount of FR in rat brains related to a photothrombotic ischemic event shortly after establishing the lesion. A model of left hemisphere photothrombosis ischemia was used in the experiment. Brains of animals from the experimental group were removed and placed in liquid N(2) for 60 min after the green laser exposure, the control group brains, exposed to the photosensitive dye Rose Bengal (RB), were placed in liquid N(2) for 80 min after RB application, naive control brains were also briefly stored in liquid N(2). Spectroscopy of electron paramagnetic (spin) resonance was used to directly measure FR (hydroxyl (OH(.)) and nitroxyl (NO(.)). Compared to naive controls, both the ischemia and RB groups had significantly higher levels of OH(.), however, there were no differences between them. Comparison of hemispheres, i.e., with and without ischemia, in the experimental group did not show any significant difference in OH(.). NO(.) were elevated in the ischemia and RB groups compare to naive controls. Higher levels of NO(.) were found in hemispheres with ischemia compared to unexposed hemispheres. Increases in OH(.) were probably associated with the action of RB itself in this model of ischemia. Increases in NO(.) were closely related to the pathogenesis of photothrombotic ischemia and could be related to the activity of nitric oxide synthases.

Oxidative stress induced by epileptic seizure and its attenuation by melatonin.

An epileptic seizure and postictal period in addition to well-known features are also characterized by massive consumption of energy. This is thought to lead to oxidative stress and increased generation of free radicals, which is reflected by increased levels of oxidative products. Our previous work described the neuroprotective effects of melatonin in preventing cognitive worsening after a single epileptic seizure. This work was aimed on direct measurement of free radicals in brain tissue using the EPR method 1, 15 and 60 minutes after seizure. The measurement was performed in adult male Wistar rats at the mentioned intervals after a single tonic-clonic seizure induced by flurothyl. In comparison to control animals there was a significant increase in hydroxyl and nitroxyl radicals 60 minutes after the seizure. The levels of hydroxyl radicals were significantly lower in animals that received melatonin 60 minutes before seizure induction compared to animals without preventive treatment. Therefore, melatonin affected the generation of the measured free radicals differently. An important finding was the delayed increase in free radicals after a single seizure in the later phases of recovery.

Stability of influenza virus as evaluated by integrity of its RNA.

UNLABELLED: Various methods of handling samples of avian influenza prior to detecting influenza viruses can significantly influence both, the detection of the virus and the quantification of viral nucleic acids. The quantity of influenza viral RNA remaining in different collecting buffers and kept at temperatures of -20°C, +4°C or +22°C for various lengths of time, was determined. The quantity of viral RNA remained the same for 120 days at -20°C, but decreased when the samples were stored at either +4°C or +22°C. The quantity of RNA was influenced by the composition of the collecting buffer. The influenza virus sample that is to be used for RNA quantification can be stored at +4°C and freeze and thaw cycles should be avoided during transport. Our results clearly indicate that the quality and quantity of influenza virus nucleic acid depends on the chemical composition of used buffer and also that the samples can be protected from degradation even if they are not stored at ultra-low temperatures. However, repeated thaw and freeze cycles will damage viral RNA even if kept in stabilizing buffers. KEYWORDS: influenza virus; degradation; RNA; buffer.

Species-specific expression of major urinary proteins in the house mice (Mus musculus musculus and Mus musculus domesticus).

The analysis of expression of pheromone-carrying major urinary proteins (MUPs) from two subspecies of house mice (Mus m. musculus, Mus m. domesticus) was studied. It has been previously shown that commensal populations of the two subspecies can discriminate on the basis of urinary signals. MUPs are predominant urinary proteins that protect pheromones from rapid degradation in a hydrophilic environment, and individuals of M. m. musculus tend to rely on these urinary cues in the process of subspecies discrimination more than M. m. domesticus individuals. Although it is not precisely known what triggers phenotypic and epigenetic changes of MUP expression, our results show that in the subspecies M. m. musculus, sex is a significant factor influencing variations in the regulation of selected MUPs in the liver. Furthermore, male M. m. musculus individuals expressed all the studied MUPs' mRNA significantly more than females or individuals of either sex in M. m. domesticus. Correspondingly, the pattern of mRNA abundance was corroborated with the level of total MUP concentration in the urine, such that the level of sexual dimorphism was also significant and species-specific. Our finding introduces a hypothesis that quantitative variation of these proteins may be an essential part of a subspecies recognition system that maintains homospecific mixing.

Iron humate as a low-cost sorbent for metal ions.

Iron humate produced as a waste by-product during an industrial manufacture of humic substances from low-rank brown coals was tested as a sorbent for the removal of metal cations--Cd (II), Cu (II), Co (II), Ni (II), Zn (II), TI (I), Eu (III), Cr (III)--as well as hexavalent chromium from waters. The Langmuir-type isotherms were used to describe the metal sorption; the respective equations may be derived and interpreted on the basis of a concept of surface-complexation reactions. Parameters of the sorption isotherms were estimated from experimental dependencies measured in a batch arrangement. The sorption capacities ranged from 0.024 mmol g(-1)for Co (II) to 0.324 mmol g(-1)for Tl(I). The metal uptake was affected by the presence of complexing agents (EDTA, salicylate, citrate), but this effect depends strongly on pH and the kind of complexing agent. The sorption of Cu (II) ions was suppressed in the presence of EDTA in an almost whole examined pH range (ca. 1-6.5) and in the presence of citrate at higher pH values (above pH 4). In the presence of salicylate, on the other hand, a slight sorption enhancement was observed. The metal sorption was suppressed also in the presence of anionic surfactant (sodium dodecylsulfate). Both in the presence as well as in the absence of complexing agents, the metal sorption showed a strong dependence on pH--the sorption was reduced considerably at low pH values below ca. 2. A typical working range for iron humate as a sorbent is ca. pH 3-5, where it exhibits a high buffering capacity, a sufficient stability and metal-binding capability.

[Oxidation stress, insulin resistance and endothelial dysfunction during the treatment of hyperlipidaemia]. / Oxidacní stres, inzulínová rezistence a endoteliální dysfunkce v prubehu lécby hyperlipidémie.

BACKGROUND: Hyperlipidaemia represents one of the major risk factors of the type 2 diabetes mellitus (DM2). In the pathogenesis of insulin resistance (IR) development glucose homeostasis impairment and their progression into DM2, oxidative stress and endothelial dysfunction (ED) may play an important role. Recent papers indicate the possibility to prevent the development of DM2 by HLP treatment, which is characterised by increased oxidation stress and ED. METHODS AND RESULTS: For the period of twelve months 46 patients with primary HLP (group S) (LDL-C > 4.1 mmol/l a TG < 3.5 mmol/l), were treated with atorvastatine 20 mg or simvastatine 40 mg. Patients with LDL-C > 4.1 mmol/l along with TG > 3.5 mmol/l were randomly divided into two groups. The SF group was treated with a combination of statin + 200 mg micronized fenofibrate each day, and group SR received together with statin a compound containing n-3 polyene fatty acids (PUFA n-3) in the daily dose of 3.6 g. After one year lasting therapy we found beside the positively influenced concentration of atherogenic lipids and lipoproteins in the group S and SF a significantly reduced concentration of conjugated dienes (CD) in LDL ( -21, resp. 16%, both P < 0.05); the test of KD kinetics in LDL in the group S has marginal increase of the lag phase (P = 0.06) and in the groups S and SR also a significant improvement of ED (increase by the flow of mediated vasodilation, FMD) by 20%, resp. by 18% (both P < 0.05) and in the SR group a significant decrease of microalbuminuria. We did not proved significant concentrations of insulin, C-peptide or indexes showing the degree of IR (HOMA and QUICKI) CONCLUSIONS: Long-lasting hypolipidemic treatment positively affected in our study the oxidative stress and ED, however, it did not resulted in changes of IR.

The effect of captopril on nitric oxide formation and on generation of radical forms of mitochondrial respiratory chain compounds in ischemic rat heart.

The increase of radical forms of mitochondrial respiratory chain compounds (MRCC) is an indicator of an increased risk of the formation of oxygen radicals. Using electron paramagnetic resonance (EPR), we found an increase of signals corresponding to ubisemichinone radical (.QH) and ironsulfur proteins radical forms (-FeS) of these respiratory chain compounds during ischemia in the isolated perfused rat heart (.QH increased from 1.51 to 3.08, .FeS1 from 1.14 to 2.65 arbitrary units). During the 5-min reperfusion, the signals returned to normoxic levels. In isolated mitochondria exposed to anoxia and reoxygenation the radical forms of .QH and FeS2 changed in a similar manner as in the intact heart. A combination of in vivo captopril treatment and in vitro L-arginine administration significantly decreased the levels of MRCC radicals in the isolated myocardium (.QH from 2.61 to 1.72 and .FeS, from 1.82 to 0.46 under normoxia; .QH from 4.35 to 2.66 and .FeS1 from 1.93 to 1.35 during ischemia). This decrease in MRCC radical forms was associated with increased NO levels in the perfusate, determined as NO2- / NO3-, as well as tissue NO levels determined using EPR as the dinitrosyl iron complex (DNIC). These results provide new information about the cardioprotective effects of ACE inhibitors and L-arginine.

Quinolinic acid-iron(ii) complexes: slow autoxidation, but enhanced hydroxyl radical production in the Fenton reaction.

Quinolinate (pyridine-2,3-dicarboxylic acid, Quin) is a neurotoxic tryptophan metabolite produced mainly by immune-activated macrophages. It is implicated in the pathogenesis of several brain disorders including HIV-associated dementia. Previous evidence suggests that Quin may exert its neurotoxic effects not only as an agonist on the NMDA subtype of glutamate receptor, but also by a receptor-independent mechanism. In this study we address ability of ferrous quinolinate chelates to generate reactive oxygen species. Autoxidation of Quin-Fe(II) complexes, followed in Hepes buffer at pH 7.4 using ferrozine as the Fe(II) detector, was found to be markedly slower in comparison with iron unchelated or complexed to citrate or ADP. The rate of Quin-Fe(II) autoxidation depends on pH (squared hydroxide anion concentration), is catalyzed by inorganic phosphate, and in both Hepes and phosphate buffers inversely depends on Quin concentration. These observations can be explained in terms of anion catalysis of hexaaquairon(II) autoxidation, acting mainly on the unchelated or partially chelated pool of iron. In order to follow hydroxyl radical generation in the Fenton chemistry, electron paramagnetic resonance (EPR) spin trapping with 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) was employed. In the mixture consisting of 100 mM DMPO, 0.1 mM Fe(II), and 8.8 mM hydrogen peroxide in phosphate buffer pH 7.4, 0.5 mM Quin approximately doubled the yield of DMPO-OH adduct, and higher Quin concentration increased the spin adduct signal even more. When DMPO-OH was pre-formed using Ti3+ /hydrogen peroxide followed by peroxide removal with catalase, only addition of Quin-Fe(II), but not Fe(II), Fe(III), or Quin-Fe(III), significantly promoted decomposition of pre-formed DMPO-OH. Furthermore, reaction of Quin-Fe(II) with hydrogen peroxide leads to initial iron oxidation followed by appearance of iron redox cycling, detected as slow accumulation of ferrous ferrozine complex. This phenomenon cannot be abolished by subsequent addition of catalase. Thus, we propose that redox cycling of iron by a Quin derivative, formed by initial attack of hydroxyl radicals on Quin, rather than effects of iron complexes on DMPO-OH stability or redox cycling by hydrogen peroxide, is responsible for enhanced DMPO-OH signal in the presence of Quin. The present observations suggest that Quin-Fe(II) complexes display significant pro-oxidant characteristics that could have implications for Quin neurotoxicity.

Does exogenous melatonin influence the free radicals metabolism and pain sensation in rat?

Melatonin has been shown to play a role in antioxidative defence. We therefore studied its effect on oxidative damage to the rat cerebral cortex evoked by painful stimulation and immobilization-induced stress. Moreover, the effect of melatonin on chronic pain perception was examined. Rats were injected with either a high dose of melatonin (100 mg/kg i.p.) or a vehicle for five days and were subjected to painful stimulation or immobilization stress 30 min after the treatment. To determine the degree of oxidative stress, the levels of free radicals, thiobarbituric acid reactive substances (TBARS) as indicators of lipid peroxidation and glutathione peroxidase (GSHPx) were estimated in somatosensory cortex. Pain perception was measured by the tail-flick and plantar test. Melatonin reduced the level of TBARS previously increased by painful stimulation. Melatonin also exhibited a slight analgesic effect in those animals exposed to painful stimulation but its role in free radical scavenging did not contribute to this effect.

Cytochromes P450 in benzene metabolism and involvement of their metabolites and reactive oxygen species in toxicity.

Cytochrome P450 (CYP) 2E1 was the most efficient CYP enzyme that oxidized benzene to soluble and covalently bound metabolites in rat and human liver microsomes. The covalent binding was due mostly to the formation of benzoquinone (BQ), the oxidation product of hydroquinone (HQ), and was inversely related to the formation of soluble metabolites. In rats, inhalation of benzene (4 mg/liter of air) caused a rapid destruction of CYP2B1 previously induced by phenobarbital. The ability of benzene metabolites to destroy liver microsomal CYP in vitro decreased in the order BQ > HQ > catechol > phenol. The destruction was reversed by ascorbate and diminished by alpha-tocopherol, suggesting that HQ was not toxic, whereas BQ and semiquinone radical (SQ) caused the effect. In the presence of nicotinamide adenine dinucleotide phosphate, reduced (NADPH) the microsomes did not oxidize HQ to BQ, while the formation of superoxide anion radical from both HQ and BQ was markedly quenched. Destruction of CYP in vitro caused by HQ or BQ was not mediated by hydroxyl radical formation or by lipid peroxidation. On the contrary, HQ and BQ inhibited NADPH-mediated lipid peroxidation. Ascorbate induced high levels of hydroxyl radical formation and lipid peroxidation, which were differentially affected by quinones, indicating different mechanisms. Despite reducing the toxicity of HQ and BQ, ascorbate appeared to induce its own toxicity, reflected in high levels of lipid peroxidation. Iron redox cycling played a significant role in the NADPH-induced hydroxyl radical formation but not in that caused by ascorbate; however, lipid peroxidation induced by NADPH or ascorbate was suppressed by ethylenediaminetraacetate, indicating a crucial role of iron. Thus, the data indicate that the quinones destroyed CYP directly and not via oxygen activation or lipid peroxidation.

The role of CYP2E1 and 2B1 in metabolic activation of benzene derivatives.

CYP2B1 and 2E1 oxidized toluene, aniline and monochlorobenzene (MCB) to water-soluble metabolites and to products covalently binding to microsomal proteins from male Wistar rats at high efficiency. Oxidation of benzene to covalently binding metabolites was catalysed by CYP2B1 and 2E1 more effectively than the formation of water-soluble metabolites, especially at low benzene levels. Thus, the formation of covalently binding products was inversely related but formation of soluble metabolites was proportional to benzene concentration. 1,4-Benzoquinone was responsible for the majority of covalent binding to microsomal proteins, being suppressed by ascorbate; 1,4-semiquinone was not important, since alpha-tocopherol did not inhibit the covalent binding and ESR showed its rapid decay, if NADPH was available. Specific antibodies and inhibitors confirmed the role of CYP2B1 and 2E1 induction. Covalent binding of benzene to DNA was largely due to benzene oxide; approximately 50% was due to N-7 guanine adduct. CYP2E1 oxidizing benzene via phenol to 1,4-hydroquinone appeared to mediate its further oxidation to 1,4-benzoquinone, which also occurred spontaneously, but was reversed in a reducing environment of microsomes with NADPH. Production of OH radicals in microsomes with NADPH was greatly stimulated by HQ and less by BQ, especially in CYP2E1 induced microsomes, although the quinones themselves failed to produce OH radicals. The quinones could act by simulation of the CYP futile cycle. Therefore, CYP2B1 and 2E1 in rats appeared essential for metabolic activation of benzene derivatives to potentially genotoxic products; BQ dominated the covalent binding of benzene to proteins, whereas DNA adducts were largely due to benzene oxide.