T. De Leo

Tissue protection against oxidative stress

We used an enhanced luminescence technique to study the response of rat tissues, such as liver, heart, muscle and blood, to oxidative stress and to determine their antioxidant capacity. As previously found for liver homogenate, the intensity of light emission (E) of tissue homogenates and blood samples, stressed with sodium perborate, is dependent on concentration, and the dose-response curves can be described by the equation E=a·C/exp(b·C). Theb value depends on the antioxidant defence capability of the tissues. In fact, it increases when homogenates are supplemented with an antioxidant, and is correlated with tissue antioxidant capacity, evaluated by two previously set up methods both using the same luminescence technique. Our results indicate that the order of antioxidant capacity of the tissues is liver>blood>heart>muscle. Thea value depends on the systems catalysing the production of radical species. In fact, it is related to the tissue level of hemoproteins, which are known to act as catalysts in radical production from hydroperoxides. The equation proposed to describe the dose-response relation is simple to handle and permits an immediate connection with the two characteristics of the systems analysed which determine their response to the pro-oxidant treatment. However, the equation which best describes the above relation for all the tissues is the following: E=α·C/exp(β·Cδ). The parameter δ assumes values smaller than 1, which seem to depend on relative amounts of tissue hemoproteins and antioxidants. The extension of the analysis to mitochondria shows that they respond to oxidative stress in a way analogous to the tissues, and that the adherence of the dose-response curve to the course predicted from the equation E=a·C/exp(b·C) is again dependent on hemoprotein content.

Free radical involvement in doxorubicin-induced electrophysiological alterations in rat papillary muscle fibres

OBJECTIVE This work was designed to determine whether the doxorubicin-induced changes in heart electrical activity are due to increased free radical production and membrane oxidative damage. METHODS Four groups of rats (60 days old) were used. One group was untreated and the others were treated with doxorubicin (DXR), DXR and vitamin E, and DXR and N-acetylcysteine (NAC), respectively. DXR was administered by single i.p. injection (20 mg/kg b.wt.). Vitamin E was administered by ten daily i.m. injections (100 mg/kg), while NAC (100 mg/kg) was injected i.p. 1 h before and 7 h after DXR. The effectiveness of the drug in inducing oxidative stress in different tissues and of the antioxidants in offering protection was established by determining antioxidant capacity, susceptibility to oxidative stress, and lipid peroxidation in heart, liver, and blood. The drug effect on heart electrical activity was determined by measuring the heart rate in vivo and action potential configuration in papillary muscle fibres in vitro. Heart lipid peroxidation and electrical activity were also examined in both vitamin E and NAC-treated rats. RESULTS DXR treatment decreased antioxidant capacity and increased lipid peroxidation and susceptibility to oxidative stress in heart and blood, but not in liver. DXR administration to rats treated with antioxidants did not produce significant changes in antioxidant capacity and susceptibility to oxidative stress even in heart and blood. Furthermore, lipid peroxidation in heart and liver from DXR- and vitamin E-treated rats, and in liver from DXR- and NAC-treated rats was lower than in untreated controls. DXR treatment also increased the duration of ventricular action potentials in untreated rats, but not in antioxidant-treated rats. The treatment of control animals with the antioxidants affected lipid peroxidation, but not cardiac electrical activity. CONCLUSIONS The protection offered by antioxidants against electrophysiological alterations indicates a free radical involvement in such alterations. In contrast, although electrical modifications are associated with increased peroxidative processes and both are prevented by the antioxidants, it is not yet clear whether a causative relationship exists between them.

Thyroid State and Mitochondrial Population during Cold Exposure

Young rats exposed to the cold (4°C) for 15–25 days exhibit remarkable modifications in their thyroid state and in the mitochondrial population of target organs such as liver.The serum total and free T3 levels more or less doubled (from 77±7 to 130±7 ng/100 ml and from 350±25 to 530 ±25 pg/100 ml, respectively) after 2 h of exposure while the serum total T4 levels underwent a limited and transitory increase; mitochondrial α-glycerophosphate dehydrogenase activity increased. On re-exposure to room temperature the thyroid state returned to normal. Cold exposure diminished the cellular volumes of hepatic cells, while the successive warm re-exposure increased the number of liver cells. The number of mitochondria per nucleus increased after 5 days of cold exposure and doubled after 10 days (from 1,200±120 to 2,400±130), while the mean protein content per organelles exhibited an exactly contrary trend.These results suggest that during cold acclimatation, the thyroid plays a role in inducing an augmentation of mitochondrial membrane surfaces per cell by stimulation of the mitochondrial protein synthesizing mechanism. At present, it is not possible to establish whether these effects are due to transcriptional modifications of the nuclear genoma only or, more likely, to a dual action at nuclear and mitochondrial level.

Effect of Thyroid State on Characteristics Determining the Susceptibility to Oxidative Stress of Mitochondrial Fractions from Rat Liver.

We have studied biochemical and functional characteristics of three mitochondrial fractions resolved by differential centrifugation from the liver of rats in different thyroid states. In particular, the relation between cytochrome and antioxidant content of the mitochondria and their oxygen consumption and response to oxidative stress has been investigated. The cytochrome content is greater in the fractions at higher density, whereas the opposite pattern is shown for antioxidant level. These biochemical characteristics seem to determine functional ones. In fact, the fraction at higher density exhibit greater oxygen consumption and smaller capacity for opposing a oxidative stress. A similar dependence of functional characteristics on biochemical ones was found in altered thyroid states. Hypothyroidism is associated with decreased cytochrome content and oxygen consumption and increased antioxidant level and effectiveness in responding to oxidative stress. The treatment of thyroidectomized rats with triiodothyronine leads to reversion of such a pattern. We are inclined to explain the differences in susceptibility to oxidative stress of the mitochondria by a modulation exerted by thyroid hormone, during the normal maturation process, on their cytochrome content. In effect, such a content is able to determine both respiratory characteristics and sensitivity to pro-oxidants of mitochondria. This sensitivity and the decrease of antioxidants, possibly due to free radical production associated with the increased oxygen flux, make the mitochondria less able to respond to an oxidative challenge.

DETERMINATION OF TISSUE SUSCEPTIBILITY TO OXIDATIVE STRESS BY ENHANCED LUMINESCENCE TECHNIQUE

Publisher Summary Tissue susceptibility to oxidation is also dependent on the activity of metal ions able to interact with oxygen species, such as the superoxide radical and hydrogen peroxide, and to produce highly reactive oxygen species such as hydroxyl radicals. This chapter presents an enhanced luminescence method that allows the evaluation of tissue susceptibility to oxidative stress and its modification in a variety of physiopathological conditions. The method has been tested on blood, liver, heart, and muscle, as well as on mitochondrial preparations from rat tissues. The method has been also used to determine the susceptibility to oxidative stress of three mitochondrial fractions resolved by differential centrifugation from rat liver in different thyroid states. It also has the advantages of being noninvasive and providing continuous monitoring.

Effect of Phenobarbital Treatment on Characteristics Determining Susceptibility to Oxidants of Homogenates, Mitochondria and Microsomes from Rat Liver

This study was designed to investigate the possible oxidative changes associated with alterations in cytochrome P450 levels in rat liver. Accordingly, extent of peroxidative processes, cytochrome and antioxidant content, capacity to face an oxidative stress were determined in liver microsomes, mitochondria, and homogenates from normal and phenobarbital (PB)-treated rats. Liver content of microsomal and mitochondrial proteins was also determined by the values of the activities of marker enzymes (glucose-6-phosphatase and cytochrome oxidase, respectively) in liver homogenate and in two cellular fractions. The increase in the liver content of microsomal and mitochondrial proteins indicated that PB caused proliferation of both smooth endoplasmic reticulum and mitochondrial population. Treatment with PB also gave rise to a general increase in peroxidative reactions (evaluated measuring malondialdehyde and hydroperoxides (HPs)), in the different cell compartments, even though HPs were not found significantly increased in mitochondrial fraction. The increase in peroxidative processes was associated with significant decreases in antioxidant concentration (expressed in terms of equivalent concentration of an antioxidant, such as the desferrioxamine), in all preparations from PB-treated rats. The response to oxidative stress in vitro (evaluated determining the parameters characterizing light emission from preparations stressed with sodium perborate) showed a substantial PB-induced increase in the susceptibility to oxidative challenge only in liver homogenate. The lack of changes in the mitochondrial preparations is likely due to decrease in concentration of both free radical producing species and antioxidants. The lack of changes in microsomal fraction is apparently in contrast with its lower oxidant capacity and higher content of cytochromes which are able to determine sensitivity to pro-oxidants. However, it could be due to the ability of cytochrome P450 to interact with the active oxygen species formed at its active center.

Modification of nucleic acid levels per mitochondrion induced by thyroidectomy or triiodothyronine administration

SummaryThe authors have determined the liver mitochondrial population (number of mitochondria/nucleus) in young rats, which has been thyroidectomized (

Antioxidant-sensitive shortening of ventricular action potential in hyperthyroid rats is independent of lipid peroxidation

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