Modulation of rat liver mitochondrial antioxidant defence system by thyroid hormone

In the present study the effect of thyroid hormone (T(3)) on oxidative stress parameters of mitochondria of rat liver is reported. Hypothyroidism is induced in male adult rats by giving 0.05% propylthiouracil (PTU) in drinking water for 30 days and in order to know the effect of thyroid hormone, PTU-treated rats were injected with 20 microg T(3)/100 g body weight/day for 3 days. The results of the present study indicate that administration of T(3) to hypothyroid (PTU-treated) rats resulted in significant augmentation of oxidative stress parameters such as thiobarbituric acid reactive substances and protein carbonyl content of mitochondria in comparison to its control and euthyroid rats. The hydrogen peroxide content of the mitochondria of liver increased in hypothyroid rats and was brought to a normal level by T(3) treatment. Induction of hypothyroidism by PTU treatment to rats also resulted in the augmentation of total and CN-sensitive superoxide dismutase (SOD) activities of the mitochondria, which was reduced when hypothyroid rats were challenged with T(3). Although CN-resistant SOD activity of the mitochondria remained unaltered in response to hypothyroidism induced by PTU treatment, its activity decreased when hypothyroid rats were injected with T(3). The catalase activity of the mitochondria decreased significantly by PTU treatment and was restored to normal when PTU-treated rats were given T(3). Total, Se-independent and Se-dependent glutathione peroxidase activities of the mitochondria were increased following PTU treatment and reduced when T(3) was administered to PTU-treated rats. The reduced and oxidised glutathione contents of the mitochondria of liver increased significantly in hypothyroid rats and their level was restored to normal when hypothyroid rats were injected with T(3). The results of the present study suggest that the mitochondrial antioxidant defence system is considerably influenced by the thyroid states of the body.     

Thyroid Hormone Influences Antioxidant Defense System in Adult Rat Brain

The objective of the current study was to find out whether thyroid hormone influences antioxidant defense parameters of rat brain. Several oxidative stress and antioxidant defense parameters of mitochondrial (MF) and post-mitochondrial (PMF) fractions of cerebral cortex (CC) of adult rats were compared among euthyroid (control), hypothyroid [6-n-propylthiouracil (PTU)-challenged], and hyperthyroid (T3-treatment to PTU-challenged rats) states. Oxidative stress parameters, such as thiobarbituric acid-reactive substances (TBA-RS) and protein carbonyl content (PC), in MF declined following PTU challenge in comparison to euthyroid rats. On the other hand, when PTU-challenged rats were treated with T3, a significant increase in the level of oxidative stress parameters in MF was recorded. Hydrogen peroxide content of MF as well as PMF of CC was elevated by PTU-challenge and brought to normal level by subsequent treatment of T3. Although mitochondrial glutathione (reduced or oxidized) status did not change following PTU challenge, a significant reduction in oxidized glutathione (GSSG) level was noticed in PMF following the treatment. T3 administration to PTU-challenged rats had no effect on mitochondrial glutathione status. Total and CN-resistant superoxide dismutase (SOD) activities in MF of CC augmented following PTU challenge. CN-resistant SOD activity did not change when PTU-challenged rats were treated with T3. Although CN-sensitive SOD activity of PMF remained unaltered in response to PTU challenge, its activity increased when PTU-challenged rats were treated with T3. Catalase activity in PMF of CC of PTU-challenged rats increased, whereas the activity was decreased when hypothyroid rats were treated with T3. Similarly, total and Se-dependent glutathione peroxidase (GPx) activities of MF increased following PTU challenge and reduced following administration of T3. Se-independent GPx activity of MF and PMF and glutathione reductase activity of PMF decreased following PTU challenge and did not change further when rats were treated with T3. On the other hand, glutathione S-transferase activity of MF and PMF of CC did not change following PTU challenge but decreased below detectable level following T3 treatment. Results of the current investigation suggest that antioxidant defense parameters of adult rat brain are considerably influenced by thyroid states of the body.     

Thyroid hormone-induced haemoglobin changes and antioxidant enzymes response in erythrocytes

Thyroid hormones modulate haemoglobin and reactive oxygen species (ROS) production, leading to antioxidant changes. This study evaluated the antioxidant response to ROS in erythrocytes in hypothyroid and hyperthyroid rats. Wistar rats were divided into four groups: control; hyperthyroid (T4-12 mg 1(-1) in drinking water); sham operated (simulation of thyroidectomy); and hypothyroid (thyroidectomized). Four weeks after, blood was collected and haemoglobin and T(4) levels, lipid peroxidation (LPO), protein oxidation, superoxide dismutase (SOD), catalase (CAT) , glutathione S-transferase (GST) and glutathione peroxidase (GPx) activities, and total radical antioxidant potential (TRAP) were measured. SOD, CAT and GST immunocontent was evaluated. Haemoglobin levels were increased in hyperthyroid erythrocytes. LPO and carbonyls were augmented (65% and 55%, respectively) in hyperthyroid and reduced (31% and 56%, respectively) in hypothyroid group. SOD and CAT activities have not changed, as well as CAT immunocontent. TRAP was diminished in both hyperthyroid and hypothyroid groups (36% and 37%, respectively). GST activity and immunocontent, as well as GPx activity, were increased in hyper and hypothyroid rats. The data suggest that thyroid hormone changes determine ROS concentration changes and decrease of some antioxidant defences that would lead to a compensatory answer of the GST and GPx enzymes, which could be consider as credible biomarkers.     

Effects of thyroid state on H2O2 production by rat heart mitochondria: sites of production with complex I- and complex II-linked substrates.

This work was designed to determine possible effects of altered thyroid states on rates and sites of H 2 O 2 production by rat heart mitochondria. Rates of O 2 consumption and H 2 O 2 release, capacities to remove the peroxide, lipid peroxidation, cytochrome oxidase activities and ubiquinone levels were determined in heart mitochondria from euthyroid, hypothyroid, and hyperthyroid rats. Hypothyroidism decreased, whereas hyperthyroidism increased the rates of O 2 consumption and H 2 O 2 release during both state 4 and state 3 respiration with Complex I- or Complex II-linked substrates. The percentage of O 2 released as H 2 O 2 was not significantly affected by thyroid state. However, the mitochondrial capacity to remove H 2 O 2 increased in the transition from hypothyroid to hyperthyroid state, which indicates that H 2 O 2 production did not modify in proportion to the rate of O 2 consumption. The thyroid-state-linked changes in H 2 O 2 production were well correlated with the levels of hydroperoxides. Rates of H 2 O 2 release in the presence of respiratory inhibitors indicated that changes in the H 2 O 2 production occurred at both sites at which H 2 O 2 was generated in euthyroid state. This result and the observation that ubiquinol levels and cytochrome oxidase activities increase in the transition from hypothyroid to hyperthyroid state suggest that the modifications of H 2 O 2 production are due to a modulation by thyroid hormone of mitochondrial content of autoxidisable electron carriers.     

Oxidative damage and antioxidant enzyme activities in experimental hypothyroidism

Free radicals are now well known to damage cellular components. To investigate whether age and thyroid level affect peroxidation speed, we examined the levels of malondialdehyde and antioxidant enzyme activities in different age groups of hypothyroid rats. Hypothyroidism was induced in 30- and 60-day-old Wistar Albino rats by the i.p. administration of propylthiouracil (10 mg kg(-1) body weight) for 15 days. While malondialdehyde levels of 30- or 60-day-old hypothyroid rats were increased in liver, they were decreased in the tissues of the heart and thyroid. While glucose-6-phosphate dehydrogenase activity levels did not change in heart, brain and liver tissues of 30-day-old rats, they increased in brain and heart tissues of 60-day-old experimental groups, but decreased in the liver. Catalase activities decreased in the liver and heart of rats with hypothyroidism, but increased in erythrocytes. In control groups while malondialdehyde levels increased in brain, heart and thymus with regard to age, they decreased in plasma. Glucose-6-phosphate dehydrogenase and catalase activities were not affected by age in tissues of the thymus, thyroid and brain, but they were decreased in the heart tissue. The changes in the levels of lipid peroxidation and antioxidant enzyme activities which were determined in different tissues of hypothyroid rats indicate a cause for functional disorder of these tissues. Moreover, there may be changes depending on age at lipid peroxidation and antioxidant enzyme activity levels.     

Tri-iodo thyronine regulates antioxidant enzyme activities in different cell fractions through a mechanism sensitive to actinomycin D in a teleost, Anabas testudineus (Bloch)

The present study evaluated the effects of hyperthyroid state on lipid peroxidation and antioxidant enzymes in the crude (CF), post nuclear (PNF) and mitochondrial fractions (MF) of the fish liver. The in vivo injection of T3 (200ng) did not change the lipid peroxidation products, malondialdehyde (MDA) and conjugated dienes (CD), while actinomycin D (10microg), a potent mRNA inhibitor when administered with T3 increased them. The antioxidant enzymes like superoxide dismutase (SOD) and catalase (CAT), glutathione peroxidase (GPx) and glutathione reductase (GR) had an increased activity in CF and MF of hyperthyroid group to compete the increased oxidative stress, but actinomycin D partially inhibited the T3-induced activity. SOD and CAT activities in PNF of hyperthyroid group had no change, the glutathione concentration varied depending on the GPx and GR activity. Hyperthyroidism decreased the protein content, while simultaneous administration of actinomycin D inhibited the T3 action of elevating the protein content. The results suggest that the antioxidant defense status in A. testudineus is modulated by thyroid hormone, through an action sensitive to actinomycin D.     

Increased resistance to hydrogen peroxide‐induced cardiac contracture is associated with decreased myocardial oxidative stress in hypothyroid rats

The purpose of this study was to determine whether decreased oxidative stress would increase the resistance to cardiac contracture induced by H₂O₂ in hypothyroid rats. Male Wistar rats were divided into two groups: control and hypothyroid. Hypothyroidism was induced via thyroidectomy. Four weeks post surgery, blood samples were collected to perform thyroid hormone assessments, and excised hearts were perfused at a constant flow with or without H₂O₂ (1 mmol/L), being divided into two sub‐groups: control, hypothyroid, control + H₂O₂, hypothyroid + H₂O₂. Lipid peroxidation (LPO) was evaluated by chemiluminescence (CL) and thiobarbituric acid reactive substances (TBARS) methods, and protein oxidation by carbonyls assay in heart homogenates. Cardiac tissue was also screened for superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) activities, and for total radical‐trapping antioxidant potential (TRAP). Analyses of SOD and glutathione‐S‐transferase (GST) protein expression were also performed in heart homogenates. Hypothyroid hearts were found to be more resistant to H₂O₂‐induced contracture (60% elevation in LVEDP) as compared to control. CL, TBARS, carbonyl, as well as SOD, CAT, GPx activities and TRAP levels were reduced (35, 30, 40, 30, 16, 25, and 33%, respectively) in the cardiac homogenates of the hypothyroid group as compared to controls. A decrease in SOD and GST protein levels by 20 and 16%, respectively, was also observed in the hypothyroid group. These results suggest that a hypometabolic state caused by thyroid hormone deficiency can lead to an improved response to H₂O₂ challenge and is associated with decreased oxidative myocardial damage. Copyright © 2009 John Wiley & Sons, Ltd.     

Significance of alterations in hepatic antioxidant enzymes. Primacy of glutathione peroxidase.

The relative contributions of catalase and the selenoenzyme glutathione peroxidase (GSH-Px) were elucidated in the rat liver by selectively modulating the activities of these enzymes using dietary selenium (Se) and the catalase inhibitor 3-amino-1,2,4-triazole (3-AT). Increased peroxidation occurred only in Se-deficient rats with markedly reduced cytosolic and mitochondrial GSH-Px activities. Although 3-AT treatment resulted in a 75% reduction of hepatic catalase activity and also a 20% reduction of both cytosolic and mitochondrial superoxide dismutase (SOD) activity, no incremental increase in peroxidation was observed over that associated with Se deficiency. In Se-deficient animals, treatment with 3-AT resulted in a doubling of cytosolic GSH-Px. This was associated with a 49% elevation in hepatic Se suggesting that increased Se may have contributed to the enhanced GSH-Px activity. These results suggest that GSH-Px plays the pivotal role in preventing hepatic peroxidation. Furthermore, the effects of 3-AT in vivo are not restricted to inhibition of catalase activity insofar as it also affects cytosolic GSH-Px activity and cytosolic and mitochondrial SOD activities.     

Serum and tissue coenzyme Q9 in rats with thyroid dysfunctions

Serum and tissue CoQ9 levels were determined in hypothyroid, euthyroid and hyperthyroid rats. A significant negative correlation was demonstrated between serum FT4 or T3 and CoQ9 in rats with various states of thyroid functions. Liver CoQ9 was significantly increased in rats rendered mildly hyperthyroid. There was a significant positive correlation between serum FT4 or T3 and liver CoQ9. While liver CoQ9 did not significantly change in severely hyperthyroid animals, liver mitochondrial CoQ9 showed a significant positive correlation with serum T3. Kidney and heart CoQ9 levels did not significantly change in hyperthyroid rats, but those in hypothyroid rats showed a tendency to increase. It was suggested that the synthesis of CoQ9 was increased in the liver in hyperthyroidism.     

Effect of thyroid state on susceptibility to oxidants and swelling of mitochondria from rat tissues

The effects of the thyroid state on oxidative damage, antioxidant capacity, susceptibility to in vitro oxidative stress and Ca(2+)-induced permeabilization of mitochondria from rat tissues (liver, heart, and gastrocnemious muscle) were examined. Hypothyroidism was induced by administering methimazole in drinking water for 15 d. Hyperthyroidism was elicited by a 10 d treatment of hypothyroid rats with triiodothyronine (10 micro g/100 g body weight). Mitochondrial levels of hydroperoxides and protein-bound carbonyls significantly decreased in hypothyroid tissues and were reported above euthroid values in hypothyroid rats after T(3) treatment. Mitochondrial vitamin E levels were not affected by changes of animal thyroid state. Mitochondrial Coenzyme Q9 levels decreased in liver and heart from hypothyroid rats and increased in all hyperthyroid tissues, while Coenzyme Q10 levels decreased in hypothyroid liver and increased in all hyperthyroid tissues. The antioxidant capacity of mitochondria was not significantly different in hypothyroid and euthyroid tissues, whereas it decreased in the hyperthyroid ones. Susceptibility to in vitro oxidative challenge decreased in mitochondria from hypothyroid tissues and increased in mitochondria from hyperthyroid tissues, while susceptibility to Ca(2+)-induced swelling decreased only in hypothyroid liver mitochondria and increased in mitochondria from all hyperthyroid tissues. The tissue-dependence of the mitochondrial susceptibility to stressful conditions in altered thyroid states can be explained by different thyroid hormone-induced changes in mitochondrial ROS production and relative amounts of mitochondrial hemoproteins and antioxidants. We suggest that susceptibilities to oxidants and Ca(2+)-induced swelling may have important implications for the thyroid hormone regulation of the turnover of proteins and whole mitochondria, respectively.     

Alleviation of iron induced oxidative stress by the grape fruit flavanone naringin in vitro

Iron is an essential element that participates in several metabolic activities of cells; however, excess iron is a major cause of iron-induced oxidative stress and several human diseases. The protective effect of naringin, a grape fruit flavanone, was studied in iron overloaded isolated mouse liver mitochondria, where the isolated mitochondrial fraction was incubated with various concentrations of naringin before ferric ion loading. Iron overloading of mitochondrial fraction resulted in an increase in lipid peroxidation, protein oxidation, and DNA damage, whereas iron overload reduced the glutathione (GSH) concentration, glutathione-S-transferase (GST), glutathione peroxidase (GSHPx), catalase and superoxide dismutase (SOD) activities. Pretreatment of mitochondrial fraction with naringin inhibited iron-induced lipid peroxidation, protein oxidation, and DNA damage. Conversely, naringin supplementation arrested iron-induced depletion in the GSH contents, GSHPx, GST, SOD and catalase activities significantly. Ferric iron reduction assay revealed that naringin could not reduce ferric iron into ferrous iron indicating that it did not exhibit prooxidant activity. Iron free coordination site assay indicated that naringin was unable to occupy all the active sites of iron indicating that naringin did not completely chelate iron. Our study demonstrates that naringin was able to share the burden of endogenous oxidants by inhibiting the iron-induced depletion of all important antioxidant enzymes as well as GSH and may act as a good antioxidant.     

Thyroid hormone-induced oxidative damage on lipids, glutathione and DNA in the mouse heart

Oxygen radicals of mitochondrial origin are involved in oxidative damage. In order to analyze the possible relationship between metabolic rate, oxidative stress and oxidative damage, OF1 female mice were rendered hyper- and hypothyroid by chronic administration of 0.0012% L-thyroxine (T₄) and 0.05% 6-n-propyl-2-thiouracil (PTU), respectively, in their drinking water for 5 weeks.

Hyperthyroidism significantly increased the sensitivity to lipid peroxidation in the heart, although the endogenous levels of lipid peroxidation were not altered. Thyroid hormone-induced oxidative stress also resulted in higher levels of GSSG and GSSG/GSH ratio. Oxidative damage to mitochondrial DNA was greater than that to genomic DNA. Hyperthyroidism decreased oxidative damage to genomic DNA. Hypothyroidism did not modify oxidative damage in the lipid fraction but significantly decreased GSSG and GSSG/GSH ratio and oxidative damage to mitochondrial DNA.

These results indicate that thyroid hormones modulate oxidative damage to lipids and DNA, and cellular redox potential in the mouse heart. A higher oxidative stress in the hyperthyroid group is presumably neutralized in the case of nuclear DNA by an increase in repair activity, thus protecting this key molecule. Treatment with PTU, a thyroid hormone inhibitor, reduced oxidative damage in the different cell compartments.     

Skeletal muscle mitochondrial free-fatty-acid content and membrane potential sensitivity in different thyroid states: involvement of uncoupling protein-3 and adenine nucleotide translocase

The effect of triiodothyronine (T3) on mitochondrial efficiency could be related to an increase in the concentrations of some proteins, such as uncoupling proteins (UCPs). Free fatty acids (FFA) seem to be a cofactor essential for the uncoupling activity of UCP3. In this paper, we report that the hypothyroidism-hyperthyroidism transition is accompanied by increases: (i) in the endogenous levels of mitochondrial FFA and (ii) in the sensitivity to FFA shown by the mitochondrial respiration rate and membrane potential, which correlated with the level of UCP3 protein. The level of the mRNA for adenine-nucleotide translocase-1 (ANT) was not affected by the thyroid state, while the ANT contribution to FFA-induced changes in mitochondrial uncoupling was low in the hypothyroid and euthyroid states but became more relevant in the hyperthyroid state at the highest concentration of FFA.     

Age-dependent variations in mitochondrial and cytosolic antioxidant enzymes and lipid peroxidation in different regions of central nervous system of guinea pigs

The age-related changes in the activities of antioxidant enzymes of mitochondrial and cytosolic fractions were measured in different regions of the central nervous system (CNS) in 10 and 32 months old guinea pigs. In old animals, the activities of superoxide dismutase (SOD) and glutathione peroxidase (GPx) were reduced (p < 0.05) in all the regions of CNS studied but catalase (CAT) declined significantly only in the cerebral cortex, hypothalamus and cerebellum. Glutathione reductase (GRd) activity declined in cerebral cortex and hypothalamus in the cytosolic fractions and only in cerebellum in the mitochondrial fraction. It is concluded that age-related decline in the activities of antioxidant enzymes is both region and enzyme specific. The endogenous lipid peroxide was found to be significantly higher (p < 0.05) in the 32 month old animals whereas, lipid peroxidation after incubating the tissue homogenate in air was found to be lower (p < 0.05). The in vitro mitochondrial lipid peroxidation decreased with age. The results indicate that accumulation of lipid peroxides takes place with ageing but the susceptibility of lipid peroxidation decreases in the older animals.     

Oxidative stress in toadfish (Halobactrachus didactylus) cardiac muscle. Acute exposure to vanadate oligomers

Vanadate solutions as ‘metavanadate’ (containing ortho and metavanadate species) and ‘decavanadate’ (containing manly decameric species) (5 mM; 1 mg/kg) were injected intraperitoneously in Halobatrachus didactylus (toadfish), in order to evaluate the contribution of decameric vanadate species to vanadium (V) intoxication on the cardiac tissue. Following short-term exposure (1 and 7 days), different changes on antioxidant enzyme activities—superoxide dismutase (SOD), catalase (CAT), selenium-glutathione peroxidase (Se-GPx), total glutathione peroxidase (GPx), lipid peroxidation and subcellular vanadium distribution were observed in mitochondrial and cytosolic fractions of heart ventricle toadfish. After 1 day of vanadium intoxication, SOD, CAT and Se-GPx activities were decreased up to 25%, by both vanadate solutions, except mitochondrial CAT activity that increased (+23%) upon decavanadate administration. After 7 days of exposure, decavanadate versus metavanadate solutions promoted different effects mainly on cytosolic CAT activity (−56% versus −5%), mitochondrial CAT activity (−10% versus +10%) and total GPx activity (+1% versus −35%), whereas lipid peroxidation products were significantly increased (+82%) upon 500 μM decavanadate intoxication. Accumulation of vanadium in total (0.137±0.011 μg/g) and mitochondrial (0.022±0.001 μg/g) fractions was observed upon 7 days of metavanadate exposure, whereas for decavanadate, the concentration of vanadium increased in cytosolic (0.020±0.005 μg/g) and mitochondrial (0.021±0.009 μg/g) fractions. It is concluded that decameric vanadate species are responsible for a strong increase on lipid peroxidation and a decrease in cytosolic catalase activity thus contributing to oxidative stress responses upon vanadate intoxication, in the toadfish heart.     

Influence of chronically altered thyroid status on the activity of liver mitochondrial glycerol-3-phosphate dehydrogenase in female inbred lewis rats.

The activity of liver mitochondrial flavoprotein-dependent glycerol-3-phosphate dehydrogenase (GPDH) is considered a reliable marker of thyroid status in acute and short-lasting experiments. The aim of this study was to ascertain whether GPDH activity could also be used as an index of thyroid status during chronic experiments over several months. We therefore analyzed GPDH activity in liver mitochondria of female inbred Lewis rats with thyroid status altered for 2 to 12 months. Hyperthyroid state was maintained by triiodothyronine (T (3)) or thyroxine (T (4)) administration, while methimazole was employed for inducing hypothyroidism. We found a seven- and three-fold increase of GPDH activity in female rats after T (3) or T (4) administration, respectively, compared to euthyroid females (8.9 +/- 2.3 nmol/min/mg protein), whereas administration of methimazole reduced the enzyme activity almost to one-third of the euthyroid values. These changes were not significantly influenced by the duration of hyperthyroid or hypothyroid treatment. We conclude that the level of the rat liver GPDH activity could serve as a useful marker for evaluation of hyperthyroid and hypothyroid status in chronic long-lasting experiments on female inbred Lewis rats.     

Rapid regulatory effect of tri-iodothyronine (T3) on antioxidant enzyme activities in a fish Anabas testudineus (Bloch): short-term in vivo and in vitro study

The short-term action of thyroid hormone tri-iodothyronine (T3) was studied in vivo and in vitro on antioxidant enzyme activities in a teleost Anabas testudineus (Bloch). T3 injection in vivo (200 ng) in normal fish decreased the lipid peroxidation products and increased superoxide dismutase (SOD), catalase and glutathione peroxidase (GPx) activities after 30 min. T3 in vitro (10(-6) M) increased the antioxidant activities of catalase, glutathione reductase (GR), GPx and glutathione level after 15/30 min, except SOD, substantiating in vivo effects in normal fish. The results suggest a rapid regulatory effect of thyroid hormone in vivo and in vitro, in the removal of reactive oxygen species in A testudineus.