Modulation of rat liver mitochondrial antioxidant defence system by thyroid hormone

Biochimica et Biophysica Acta 1537 (2001) 1^13 www.bba-direct.com

Modulation of rat liver mitochondrial antioxidant defence system by thyroid hormone Kajari Das, G.B.N. Chainy * Biochemistry Unit, Department of Zoology, Utkal University, Bhubaneswar 751 004, India Received 14 July 2000; received in revised form 5 April 2001; accepted 5 April 2001

Abstract In the present study the effect of thyroid hormone (T3 ) 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 Wg T3 /100 g body weight/day for 3 days. The results of the present study indicate that administration of T3 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 T3 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 T3 . 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 T3 . The catalase activity of the mitochondria decreased significantly by PTU treatment and was restored to normal when PTU-treated rats were given T3 . Total, Se-independent and Se-dependent glutathione peroxidase activities of the mitochondria were increased following PTU treatment and reduced when T3 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 T3 . The results of the present study suggest that the mitochondrial antioxidant defence system is considerably influenced by the thyroid states of the body. ß 2001 Elsevier Science B.V. All rights reserved. Keywords: Liver; Mitochondria; 3,3P,5-Triiodothyronine; Oxidative stress; Antioxidant enzyme; Rat

1. Introduction The tetravalent electron reduction of molecular oxygen to water is one of the important reactions which take place in the terminal end of the mito-

* Corresponding author. Fax: +91-674-50-90-37. E-mail address: [email protected] (G.B.N. Chainy).

chondrial electron transport chain during cellular respiration and it is considered essential for the existence of living organisms [1]. Nevertheless, the reduction of oxygen molecule in the electron transport chain is a double sword because about 1^2% of the total oxygen consumed by mitochondria during respiration is incompletely reduced and forms superoxide radicals at various sites of the electron transport chain [2]. If superoxide radicals are not immediately neutralised, they are converted to other reactive oxy-

0925-4439 / 01 / $ ^ see front matter ß 2001 Elsevier Science B.V. All rights reserved. PII: S 0 9 2 5 - 4 4 3 9 ( 0 1 ) 0 0 0 4 8 - 5

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gen species (ROS) such as hydroxyl radical and hydrogen peroxide in the presence of iron by a classical Fenton reaction [3]. Consequently, mitochondria are considered as the richest source of ROS [4]. Due to their high reactivity and non-speci¢c nature reactive oxygen species can attack almost all biomolecules present in their vicinity including membrane lipids causing oxidative stress in cells [3]. However, the cellular system is equipped with various enzymes and antioxidant small molecules, which e¡ectively neutralise ROS and protect cells and their organelles including mitochondria from deleterious e¡ects of ROS. Superoxide dismutase (Cu-Zn superoxide dismutase in case of cytoplasm and Mn-superoxide dismutase in case of mitochondria, SOD; EC 1.15.1.1) e¡ectively dismutates superoxide radicals into hydrogen peroxide which in turn is neutralised by glutathione peroxidase (GPx; EC 1.11.1.9) and catalase (CAT; EC 1.11.1.6) [3]. Small antioxidant molecules such as vitamin C, vitamin E [5,6], reduced glutathione (GSH) [7] and ubiquinol-10 [8,9] also play a crucial role in scavenging ROS. Thyroid hormones are known to in£uence several mitochondrial functions including oxygen consumption [10], oxidative phosphorylation [11], proton leak [12] and its biogenesis [13]. It will not be out of context to mention that consumption of oxygen by mitochondria is greatly in£uenced by their respiratory state, for example it is considerably higher in state 3 than in state 4 [11]. Nevertheless, the generation of ROS in mitochondria is more in state 4 than in state 3 [4]. Therefore, ROS production in mitochondria is not a simple re£ection of their respiratory state. Although there are many reports which clearly demonstrated modulation of the antioxidant defence system of tissues by the thyroid state of the body [14^18], the e¡ect of thyroid hormones on mitochondrial antioxidant capacity is not available. Against this background, the present investigation was designed to evaluate the changes in antioxidant enzymes and non-enzymatic antioxidant small molecules in mitochondria of rat liver in hypo- and hyperthyroid state with an attempt to construct a composite biochemical picture for clarifying the role of thyroid hormone in mitochondrial antioxidant capacity.

2. Materials and methods 2.1. Chemicals 6-n-Propyl-2-thiouracil (PTU) was purchased from E. Merck (Mumbai, India). 3,3P,5-Triiodothyronine (T3 ), thiobarbituric acid (TBA), catalase, glutathione reductase, cumene hydroperoxide, Sephadex G-25 and 5,5P-dithiobis-2-nitrobenzoic acid (DTNB) were obtained from Sigma (St. Louis, MO, USA). tertButyl hydroperoxide and EDTA were purchased from Merck-Schudant (Germany). Horseradish peroxidase (HRP), NADPH, reduced and oxidised glutathione (GSH and GSSG), L-methionine, ribo£avin, guanidine hydrochloride and DNPH were obtained from SISCO Research Laboratory (India). Hydroxylamine hydrochloride was purchased from KochLight Laboratory (UK). 2.2. Animals Adult male Wistar strain rats aged 90^120 days and weighing about 350^400 g were obtained from the National Institute of Nutrition (Hyderabad, India) and used in the present study. The animals were acclimatised in the animal room of the department for at least 10 days prior to experimentation. They were fed with freshly prepared food containing wheat (Triticum sativum, 40%), gram (Cicer arietinum, 20%) and rice £our (Oryza sativa, 30%), vitaminised skimmed milk powder (5%), re¢ned vegetable oil (3%), egg (1%) and common salt (1%) in the morning and water soaked whole gram in the afternoon. Tap water was supplied ad libitum. 2.3. Treatments Rats were rendered hypothyroid by the administration of 0.05% 6-n-propyl-2-thiouracil in their drinking water for 30 days [19]. Hyperthyroidism was induced in rats by one daily intraperitoneal injection of 20 Wg T3 /100 g body weight for 3 consecutive days in 0.1 mM NaOH solution [13] to PTUtreated rats. Control rats received an equal volume of vehicle solution only.

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2.4. Preparation of subcellular fractions Rats were killed by decapitation and liver was immediately dissected out, cleaned in ice-cold normal saline, pat dried on ¢lter paper and kept in ice. Liver was homogenised (10%, w/v) in ice-cold 0.25 M sucrose^phosphate bu¡er solution (50 mM, pH 7.4) in a motor-driven homogeniser. The mitochondrial fraction was obtained by the di¡erential centrifugation method as described earlier [20]. The purity of the mitochondrial fraction was assessed by estimating succinate dehydrogenase activity [21]. Crude homogenate, mitochondrial and postmitochondrial fractions were processed immediately for various biochemical analyses. 2.5. Estimation of protein The protein content of the various samples was estimated according to the method of Lowry et al. [22]. 2.6. Estimation of endogenous and induced lipid peroxidation The lipid peroxidation level in crude homogenate (10%, w/v homogenate in 50 mM PO4 bu¡er pH 7.4) and in the mitochondrial fraction was quantitated. To check the susceptibility of thiobarbituric acid reactive substances (TBARS) in mitochondria of rat liver to various oxidants in di¡erent thyroid states, mitochondria obtained from di¡erent thyroid states were incubated with H2 O2 (600 nmol/mg protein), tert-butyl hydroperoxide (t-BuOOH, 600 nmol/mg protein) and ferrous sulphate/ascorbic acid (0.25 mM/0.01 mM) [23]. Endogenous as well as oxidantinduced lipid peroxidation was quantitated by monitoring the formation of TBARS according to the method of Ohkawa et al. [24] in the presence of 0.02% w/v butylated hydroxytoluene (BHT) in the TBA reagents to suppress peroxidation during the assay itself. The concentration of TBARS was calculated from its extinction coe¤cient of 1.56U1035 M31 cm31 [25]. 2.7. Estimation of hydrogen peroxide The hydrogen peroxide content in mitochondrial

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and postmitochondrial fractions was determined spectrophotometrically according to the method of Pick and Keisari [26] using horseradish peroxidase and phenol red. 2.8. Estimation of protein carbonyl The protein carbonyl content in the mitochondrial fraction was estimated according to the method described by Levine et al. [27]. 2.9. Estimation of reduced glutathione, mitochondrial total glutathione and protein thiol content Rat liver mitochondria were prepared in a medium containing 0.25 M sucrose, 5 mM HEPES bu¡er and 1 mM EDTA, pH 7.2 (30 mg/ml). Mitochondrial samples were precipitated in ice-cold 5% trichloroacetic acid containing 0.01 N HCl and subjected to centrifugation at 1000Ug for 15 min. One part of the supernatant was used for estimation of reduced, oxidised and total glutathione with minor modi¢cations [28,29]. The colour intensity was measured 30 min after DTNB addition and GSH concentration was calculated by taking vA412 mW/30 min. The other part of the supernatant and protein precipitate were used to measure non-protein thiol and protein thiol contents respectively by the methods of Wudarczyk and Sedlak [30,31] with minor modi¢cations. 2.10. Estimation of activities of antioxidant enzymes A 10% tissue homogenate was prepared in ice-cold 0.25 M sucrose phosphate bu¡er (50 mM, pH 7.4) and centrifuged at 900Ug for 5 min to precipitate nuclei and cellular debris. The supernatant was centrifuged at 10 000Ug for 20 min at 4³C to separate the mitochondrial pellet. The mitochondrial pellet was washed with ice-cold sucrose phosphate bu¡er solution three times and dissolved in the same bu¡er (1 mg protein/0.1 ml). Prior to assay the mitochondrial fraction was freeze thawed three times. The postmitochondrial fraction was used directly for assay of catalase activity according to the method of Cohen et al. [32]. One millilitre of postmitochondrial supernatant and freeze thawed mitochondrial fraction were passed through a 5 ml column of Sephadex

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G-25 and elutes were used for the estimation of the activities of the di¡erent enzymes (SOD, GPx, GR, and GST). Superoxide dismutase was estimated by a modi¢ed nitrite method [33] which involves generation of superoxide radical by photoreduction of ribo£avin [34] and its detection by nitrite formation from hydroxylamine hydrochloride at 543 nm using Greiss reagent [35,36]. Glutathione peroxidase, glutathione reductase (GR; EC1.6.4.2) and glutathione S-transferase (GST; EC 2.5.1.18) activities were measured according to the method of Pagila and Valentine [37], Massey and Williams [38] and Habig et al. [39] respectively. Cu-Zn SOD was distinguished from Mn-SOD by incubating samples with 5 mM KCN (¢nal concentration). Total GPx activity was measured taking cumene hydroperoxide as substrate whereas Se-dependent enzyme activity was assayed taking t-BuOOH as substrate. 2.11. Statistics All data were subjected to one-way analysis of ANOVA followed by Duncan's New Multiple Range Test to determine the level of signi¢cance between control and treatment means. A di¡erence was considered signi¢cant at the P 6 0.05 level.

Table 1 E¡ect of various oxidants on TBARS (nmol/mg protein) formation of the mitochondrial fraction of liver from euthyroid, hypothyroid and hypothyroid+T3 rats Condition

nmol TBARS produced/mg protein euthyroid

No oxidant H2 O2 t-buOOH FeSO4 /AA

a

2.6 þ 0.15 2.41 þ 0.16a 6.27 þ 0.56b 7.56 þ 0.70b

hypothyroid a

2.39 þ 0.31 2.39 þ 0.15a 5.20 þ 0.42b 5.60 þ 0.21b

hypothyroid+T3 3.71 þ 0.30c 3.6 þ 0.21c 10.40 þ 2.20d 9.38 þ 1.51d

Values are mean þ S.D. of ¢ve animals. Superscripts of di¡erent letters di¡er signi¢cantly (P 6 0.05) from each other.

3. Results 3.1. Body weight and protein content (Fig. 1) The body weight of PTU-treated hypothyroid rats was signi¢cantly low in comparison to the respective control group which did not change much when rats were treated with T3 for a period of 3 days (data not given). Although a signi¢cant decrease in protein content of the mitochondrial fraction of rat liver was noticed following PTU treatment for 30 days, no signi¢cant change was noticed in case of crude homogenate (Fig. 1). No change in protein content

Fig. 1. E¡ect of thyroid hormone T3 on protein content of postmitochondrial fraction (PMF) and mitochondrial fraction (MF) of rat liver. Left, control; middle, control+PTU; right, control+PTU+T3 . Values are mean þ S.D. of six animals. Superscripts of di¡erent letters di¡er signi¢cantly (P 6 0.05) from each other.

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Fig. 2. E¡ect of thyroid hormone T3 on lipid peroxidation (TBARS) of crude homogenate (CH) and mitochondrial fraction (MF) of rat liver. Left, control; middle, control+PTU; right, control+PTU+T3 . Values are mean þ S.D. of six animals. Superscripts of di¡erent letters di¡er signi¢cantly (P 6 0.05) from each other.

was observed when T3 was administered to PTUtreated rats for a period of 3 days. 3.2. Endogenous and oxidant-induced lipid peroxidation (Fig. 2, Table 1) The lipid peroxidation level expressed in terms of

TBARS in crude homogenate as well as mitochondrial fraction of rat liver did not change signi¢cantly in response to PTU treatment from that of the control rats. However, T3 treatment of PTU-treated rats resulted in an elevation of its level in both crude homogenate and mitochondrial fraction in comparison to PTU-treated and control rats (Fig. 2). In all

Table 2 (A) E¡ect of thyroid hormone T3 on protein-SH and non-protein ^SH contents (nmol thiols/mg mitochondrial protein) of mitochondria of rat liver Parameter

nmol/mg mitochondrial protein euthyroid

Protein-SH Non-protein ^SH (GSH)

hypothyroid a

44.62 þ 0.87 5.57 þ 0.498a

b

48.60 þ 2.56 10.86 þ 0.50b

hypothyroid+T3 28.61 þ 2.93c 3.28 þ 0.19c

(B) E¡ect of thyroid hormone T3 on total, oxidised and reduced glutathione (nmol thiols/mg mitochondrial protein) and GSH/GSSG ratio of mitochondria of rat liver Parameter

nmol/mg mitochondrial protein euthyroid

Total GSH-eq GSSG GSH GSH:GSSG

hypothyroid a

12.70 þ 0.95 0.40 þ 0.07a 11.92 þ 1.04a 30.98 þ 6.83a

b

34.54 þ 0.74 2.59 þ 0.2b 29.36 þ 0.79b 11.41 þ 1.05b

hypothyroid+T3 10.84 þ 0.71c 0.37 þ 0.01a 10.11 þ 0.69a 27.70 þ 1.35a

Values are mean þ S.D. of ¢ve animals. Superscripts of di¡erent letters di¡er signi¢cantly (P 6 0.05) from each other.

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Fig. 3. E¡ect of thyroid hormone T3 on H2 O2 content of postmitochondrial fraction (PMF) and mitochondrial fraction (MF) of rat liver. Left, control; middle, control+PTU; right, control+PTU+T3 . Values are mean þ S.D. of six animals. Superscripts of di¡erent letters di¡er signi¢cantly (P 6 0.05) from each other.

the states of the thyroid, i.e. euthyroid, hypothyroid and hyperthyroid, incubation of the mitochondrial fraction with H2 O2 failed to induce formation of TBARS. In vitro incubation of liver mitochondria with t-BuOOH and ferrous iron/ascorbate (AA) showed susceptibility to lipid peroxidation, the values of which were di¡erent for mitochondria of different thyroid states. The induction of formation of TBARS by both oxidants was very low in case of PTU-treated rats when compared to control rats. The induction of formation of TBARS in mitochondria of T3 -treated rats was very high by t-BuOOH compared to that of control. However, FeSO4 /AAmediated induction was lower in T3 -treated mitochondria than in control mitochondria. 3.3. Hydrogen peroxide (Fig. 3A,B) PTU treatment in rats resulted in the elevation of the hydrogen peroxide level in both mitochondrial and postmitochondrial fractions of liver. Administra-

tion of T3 to PTU-treated rats resulted in a signi¢cant decrease in its level in both fractions in comparison to PTU-treated and control groups. 3.4. Protein carbonyl (Fig. 3C) The protein carbonyl content was measured in the mitochondrial fraction of rat liver. It is observed that PTU treatment of rats had no e¡ect on the protein carbonyl content of the mitochondrial fraction. A signi¢cant elevation in its level in the mitochondrial fraction was noticed following T3 treatment of PTUtreated rats. 3.5. Reduced glutathione, oxidised glutathione and protein thiol contents (Table 2) Both GSH content and protein-SH were found to be elevated in mitochondria of hypothyroid rat liver and were seen to be depleted below the control level after T3 treatment of PTU-treated rats (Table 2A).

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Fig. 4. E¡ect of thyroid hormone T3 on SOD activities of postmitochondrial fraction (A) and mitochondrial fraction (B) of rat liver. Left, control; middle, control+PTU; right, control+PTU+T3 . Values are mean þ S.D. of six animals. Superscripts of di¡erent letters di¡er signi¢cantly (P 6 0.05) from each other.

The enzymatic recycling assay of GSH:GSSG also evidenced a similar result in total glutathione equivalent (GSH+GSSG), oxidised glutathione (GSSG) as well as reduced glutathione (GSH) contents. However, the GSH/GSSG ratio was found to be declined in hypothyroid condition and was elevated to its control level after T3 treatment (Table 2B). 3.6. Superoxide dismutase (Fig. 4) Treatment of rats with PTU resulted in the elevation of total and CN-sensitive SOD activities in mitochondrial and postmitochondrial fractions of liver (Fig. 4). The treatment had no signi¢cant e¡ect on CN-resistant SOD activity of the mitochondrial fractions (Fig. 4B). Administration of T3 to PTU-treated rats resulted in a decrease in total and CN-sensitive SOD activities in mitochondrial (Fig. 4B) and postmitochondrial fractions (Fig. 4A) of liver in comparison to PTU-treated rats. Similarly, a signi¢cant decrease in CN-resistant SOD activity of mitochondria was recorded in response to T3 treatment of PTUtreated rats (Fig. 4B). 3.7. Catalase (Fig. 5) Treatment of rats with PTU resulted in a signi¢-

Fig. 5. E¡ect of thyroid hormone T3 on CAT activities of postmitochondrial fraction (PMF) and mitochondrial fraction (MF) of rat liver. Left, control; middle, control+PTU; right, control+PTU+T3 . Values are mean þ S.D. of six animals. Superscripts of di¡erent letters di¡er signi¢cantly (P 6 0.05) from each other.

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Fig. 6. E¡ect of thyroid hormone T3 on total (T), selenium-dependent (Se-D), and selenium-independent (Se-I) GPx activities of postmitochondrial fraction (PMF) and mitochondrial fraction (MF) of rat liver. Left, control; middle, control+PTU; right, control+PTU+T3 . Values are mean þ S.D. of six animals. Superscripts of di¡erent letters di¡er signi¢cantly (P 6 0.05) from each other.

cant decrease in catalase activity in mitochondrial and postmitochondrial fractions of liver. A signi¢cant increase in its level in mitochondrial and postmitochondrial fractions of liver of PTU+T3 -treated rats was noticed in comparison to PTU-treated rats. 3.8. Glutathione peroxidase (Fig. 6) Total and selenium-independent GPx activities in postmitochondrial fractions of rat liver increased following PTU treatment (Fig. 6A). PTU had no e¡ect on the selenium-dependent (Se-D) GPx activity of the postmitochondrial fraction of rat liver. Administration of T3 to PTU-treated rats resulted in a decrease in total and selenium-dependent GPx activity in postmitochondrial fractions. On the contrary, a signi¢cant elevation in Se-I GPx activity in the postmitochondrial fraction of liver of PTU-treated rats was noticed following T3 treatment (Fig. 6A). In the mitochondrial fraction also a signi¢cant elevation in activities of total, Se-D and Se-I GPx was noticed following PTU treatment (Fig. 6B). Activities of total, Se-D and Se-I GPx in the mitochondrial fraction of liver of PTU-treated rats were reduced signi¢cantly by T3 treatment (Fig. 6B).

Fig. 7. E¡ect of thyroid hormone T3 on glutathione reductase activity of postmitochondrial fraction (PMF) of rat liver. Left, control; middle, control+PTU; right, control+PTU+T3 . Values are mean þ S.D. of six animals. Superscripts of di¡erent letters di¡er signi¢cantly (P 6 0.05) from each other.

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Fig. 8. E¡ect of thyroid hormone T3 on glutathione S-transferase activity of postmitochondrial fraction (PMF) and mitochondrial fraction (MF) of rat liver. Left, control; middle, control+PTU; right, control+PTU+T3 . Values are mean þ S.D. of six animals. Superscripts of di¡erent letters di¡er signi¢cantly (P 6 0.05) from each other.

3.9. Glutathione reductase (Fig. 7) Glutathione reductase activity in the postmitochondrial fraction of rats decreased following PTU treatment. The activity of the enzyme further decreased in PTU-treated rats when they were treated with T3 . 3.10. Glutathione S-transferase (Fig. 8) PTU treatment resulted in a signi¢cant elevation in glutathione S-transferase activity in mitochondrial and postmitochondrial fractions of rat liver. Administration of T3 to PTU-treated rats resulted in a decrease in the enzyme activity in mitochondrial and postmitochondrial fractions of liver. 4. Discussion In order to clarify the role of thyroid hormone on the antioxidative capacity of the mitochondria of rat liver, several biochemical parameters related to oxi-

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dative stress are compared in mitochondria of liver of normal (euthyroid), PTU-treated (hypothyroid) and PTU+T3 -treated rats. The achievement of a hypothyroid state in rats by providing 0.05% PTU in drinking water for a period of 1 month is well established by several workers [16,19]. In order to know whether the changes in mitochondrial parameters are due to suppression of the thyroid level in the body, PTU-treated rats were injected with 20 Wg/100 g body weight T3 for 3 consecutive days. The decrease in body weight and tissue protein content following PTU treatment con¢rms the induction of hypothyroidism in rats and is in agreement with earlier ¢ndings [17]. PTU is reported to prevent oxidation of iodide to iodine and blocks the formation of T4 and T3 . In all vertebrate species the principal secretory product of the thyroid gland is T4 and conversion of T4 to T3 takes place in peripheral tissues. Lipid peroxidation and protein carbonyl content, markers of oxidative stress [40], did not show any change following hypothyroidism; nevertheless, both parameters increased compared to the euthyroid state when T3 was administered to hypothyroid rats. The change in lipid peroxidation in mitochondria may be due to the signi¢cant change in oxidative metabolism in mitochondria by the altered thyroid state of the body. It has been reported that respiratory activities of rat liver mitochondria are reduced following PTU treatment and escalated in response to T3 administration [14,41,42]. Our results on lipid peroxidation in crude homogenate are also corroborative with previous studies where 3 days of T3 treatment of rats resulted in a signi¢cant enhancement of lipid peroxidation in rat liver [18,43,44]. Earlier reports suggest that lipid peroxidation increase is a consequence of an increase in the degree of unsaturation of fatty acid in tissues [45^47] and thyroid hormone has a considerable in£uence on mitochondrial fatty acid composition [48]. Further, to ¢nd out whether an alteration in the thyroid state has any in£uence on the mitochondrial peroxidation capacity, the sensitivity of mitochondrial fractions of di¡erent thyroid states to in vitro peroxidation was assessed by challenging mitochondrial samples with various oxidants such as H2 O2 , t-BuOOH and FeSO4 /AA. Incubations were done under identical conditions and the levels of TBARS were estimated in all the three states of the thyroid, i.e. euthyroid, hypo-

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thyroid and hyperthyroid. Incubation of mitochondrial fractions with H2 O2 failed to induce formation of TBARS. This is not surprising if one considers the fact that the partially puri¢ed mitochondrial fractions are usually contaminated with peroxisomes, a major source of catalase enzyme which breaks down H2 O2 . It is an established fact that except in rat heart, catalase activity is usually absent in mitochondria of most of the tissues of rats including liver. In fact, incubation of the mitochondrial fraction of aminotriazole-pretreated rats with H2 O2 failed to induce formation of TBARS. Aminotriazole is known to be a potent inhibitor of catalase [23]. The extent of induction of TBARS by t-BuOOH and FeSO4 /AA in the mitochondrial fraction of the liver of hypothyroid rats is low in comparison to that in control rats. This suggests that the total peroxidation capacity of liver mitochondria is in£uenced by the thyroid state of the body. It has been reported that incubation of T3 -treated rat liver mitochondria with t-BuOOH resulted in the augmentation of ROS production [15]. The observed augmentation of peroxidation in the mitochondrial fraction of T3 -treated rats by oxidants corroborates the above statement. Our results are not only in accordance with the above ¢nding but also suggest that the production of ROS in response to oxidants is modulated by the thyroid status of the body, con¢rming a critical role of thyroid hormone in mitochondrial ROS metabolism. Similarly, the protein carbonyl content of mitochondria of rat liver was also enhanced in response to T3 treatment. It has been reported that the levels of glycation and oxidation products of liver proteins are considerably increased in the hyperthyroid state [17]. One of the interesting observations of the present study is the dramatic e¡ect of thyroid hormone on the hydrogen peroxide level of mitochondria. The hypothyroid state resulted in an increase in the mitochondrial hydrogen peroxide level which decreased to less than in the euthyroid state when animals were challenged with T3 . Thus, it is apparent that hydrogen peroxide production in mitochondria is under tight control of thyroid hormone. Since the increase and decrease in hydrogen peroxide level in the postmitochondrial fraction following an altered thyroid state are more or less similar to that of the mitochondrial fraction, it can be concluded that the regulation of the hydrogen peroxide level in both

mitochondrial and postmitochondrial fractions is more or less similar. However, the e¡ect of the hormone is more pronounced in case of the postmitochondrial fraction. Hydrogen peroxide is formed in the cellular system due to dismutation of superoxide radicals by the enzyme superoxide dismutase and reduction of H2 O2 is e¡ectively catalysed by catalase and glutathione peroxidase enzymes [3]. It is observed that hypothyroidism induced by PTU treatment resulted in the augmentation of the SOD level both in postmitochondrial and mitochondrial fractions, which is accompanied with a decrease in catalase activity. SOD activity reduced and CAT activity increased following T3 administration to PTUtreated rats. Such a change in the levels of both enzymes may be responsible for the change in hydrogen peroxide level in postmitochondrial and mitochondrial fractions. It is apparent from the present study that SOD and CAT, the two principal enzymes responsible for the metabolism of hydrogen peroxide in liver, are under the regulatory in£uence of the thyroid status of the body. It will not be out of context to mention here that catalase is not a mitochondrial enzyme in the liver of rat. It is mainly present in peroxisomes. Therefore, the possibility of contamination of peroxisomes with mitochondria and a change in their quantity may be the reason for the observed variation in catalase activity of the mitochondria. The rate of mitochondrial superoxide radical and hydrogen peroxide generation is the highest under state 4 respiration (excess substrate but no ADP) in the coupled mitochondria [4]. The hypothyroid state drastically reduced state 4 respiration which is reversed following T3 exposure [11]. If this is the case one would expect less hydrogen peroxide in mitochondria of hypothyroid rats and more in T3 -administered PTU-treated rats. Thus the most probable explanation for the observed negative correlation between hydrogen peroxide content in mitochondria and thyroid state is the alteration in hydrogen metabolising enzymes in mitochondria by the hormone. This interpretation is supported by the fact that SOD and catalase, two enzymes associated with hydrogen peroxide formation and breakdown, are considerably modulated by the thyroid state of the body. An increase in SOD activity in the hypothyroid state will accelerate the production of hydrogen peroxide while a decrease in catalase activity will slow down its re-

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moval. It is reported that production of superoxide radicals leads to the inactivation of catalase activity and the consequent accumulation of hydrogen peroxide causes inactivation of SOD [49]. In the present study, the elevated hydrogen peroxide level in the liver of the hypothyroid state is neither accompanied with a decrease in SOD activity nor with elevated lipid peroxidation. On the contrary, the hypothyroid state is associated with an elevation in SOD activity and an unchanged lipid peroxidation level in the liver. It is yet to know the physiological signi¢cance of the elevated H2 O2 level following hypothyroidism. There are two types of SOD enzymes reported in higher vertebrates. One is Cu-Zn SOD, mainly found in the cytoplasm of cells, while the other one is mitochondrial in nature and is known as Mn-SOD [50,51]. The activity of Cu-Zn SOD is highly susceptible to CN3 while Mn-SOD is resistant to CN3 inactivation [49]. Therefore, CN3 is used to distinguish the two enzyme activities from each other. In the present study, while postmitochondrial CN-sensitive SOD increased following hypothyroidism, no such change is recorded for CN-resistant SOD of mitochondria. But both CN-sensitive SOD in the postmitochondrial fraction and CN-resistant SOD in the mitochondrial fraction were inhibited by T3 treatment of hypothyroid rats, suggesting a similar regulation of both isoenzymes by thyroid hormone. The observed diminution of SOD activity in liver of rats following hyperthyroidism can be correlated with the observations of Simon et al. [52]. However, our results are not in good agreement with the ¢ndings of Seymen et al. [53], who noticed that hyperthyroidism induced in rats by T3 caused an elevation of SOD activity in liver. Such a discrepancy between our and their results may be due to di¡erent experimental conditions and di¡erent methods used to assay SOD activities. But the decrease in CN-sensitive SOD in postmitochondrial supernatant and CN-resistant SOD in mitochondria of treated rats suggests that T3 has a negative regulatory e¡ect on the expression of SOD enzyme but a positive regulatory e¡ect on CAT activity. GSH is a tripeptide widely distributed and involved in many biological activities including neutralisation of ROS, detoxi¢cation of xenobiotics, and maintenance of ^SH levels in proteins [7]. Both reduced and oxidised glutathione levels were elevated

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in the mitochondria of liver following hypothyroidism. The levels were restored to their normal values in response to T3 administration to PTU-treated rats. This con¢rms a critical role of thyroid hormone in maintaining the GSH pool in the mitochondria of liver. Distinct pools of GSH are found in cytoplasm and mitochondria. Previous studies have con¢rmed that little or no de novo synthesis of GSH occurs in the mitochondria [54]. It is an established fact that almost all cellular GSH are synthesised in the cytoplasm of liver. Hence, an increased mitochondrial GSH pool must have derived from the cytoplasm. The increase in GSH content in liver under the hypothyroid state may be an adaptive response to protect the mitochondria from the elevated level of H2 O2 . GSH is reported to be involved in numerous mitochondrial functions including mitochondrial membrane structure and integrity, ion homeostasis and mitochondrial redox state activity of numerous ^ SH-dependent enzymes [55]. The increase in the GSH level in mitochondria of hypothyroid rats may give protection to ^SH-dependent proteins. In fact, the level of the increase in protein-SH groups in the hypothyroid state corroborates the above statement. GSH:GSSG in tissue is now considered one of the important markers of oxidative stress. The decrease in its ratio and the restoration to its normal value by T3 administration again con¢rms the critical role of thyroid hormone in regulating mitochondrial oxidative stress. In mammalian system, low molecular weight isoenzymes of GST exhibit a similar activity as that of Se-independent GPx which metabolises organic hydroperoxides [55]. Both GST and Se-independent GPX activities of mitochondria and postmitochondrial fractions increased following the hypothyroid state, whereas the increased GST activity in both fractions of the hypothyroid state is brought to a low level by T3 supplementation. Only mitochondrial Se-independent GPx activity was reduced by the hormone treatment. The results of the present study indicate that the regulation of glutathione metabolism in mitochondria is also under the control of thyroid hormone. In summary, one can conclude that under normal conditions there exists a delicate balance between the rate of formation of reactive oxygen species and the rate of breakdown of reactive oxygen species in mi-

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tochondria, which is under the subtle control of thyroid hormone. Any alteration in the thyroid state of the body will considerably in£uence the antioxidative status of mitochondria and can lead to a pathophysiological state. The present study also suggests that the modulation of the mitochondrial oxidative capacity by T3 is mediated through antioxidant metabolising enzymes.

[13]

[14]

[15]

Acknowledgements We are thankful to the Head, Department of Zoology, Utkal University, Bhubaneswar for providing laboratory facilities and the Department of Science and Technology and Department of Biotechnology, Govt. of India, and UGC for ¢nancial assistance.

[16]

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