Relationship between the Dynamics of Oxidative Stress and Thyroid State

 

1Kale MK*1Bhusari KP and 2Umathe SN

 

1Sharad Pawar College of Pharmacy, Wanadongri, Hingna Road, Nagpur

2Department of Pharmaceutical Sciences, R.T.M. Nagpur University, Amaravati Road, Nagpur.

*Corresponding Author E-mail: kalemkncp@rediffmail.com

 

ABSTRACT

To examine the possible relationship between the dynamics of oxidative stress and thyroid state to evaluate role of thyroxine in oxidative stress. The Sprague-Dawley rats of either sex were equally divided in to three groups: control, hypothyroid, and hyperthyroid. Hypothyroidism was induced by administering methimazole in drinking water (0·02% w/v) for 15 days. Hyperthyroidism was induced by daily i.p. administration of tri-iodothyronine (T3) (100 µg/kg body weight) for 10 days in methimazole treated rats to induce mild hyperthyroidism in hypothyroid rats. At the end of the protocol, blood was withdrawn from retro-orbital plexus. The parameters like lipid peroxidation, superoxide dismutase and catalase were determined in erythrocyte lysate while reduced glutathione was determined in blood to find out the extent of oxidative stress.  Same blood was utilized for testing of thyroid function by determining the levels of T3, T4 & TSH. We found suppressed TSH level, with elevation of T4 and T3, increased lipid peroxidation and decreased superoxide dismutase, catalase and glutathione in hyperthyroid, as observed in oxidative stress. These were reversed with methimazole, an antithyroid in hypothyroid rats.  Hence, there exist some relationship between the dynamics of oxidative stress and thyroid state.  Reports as well as our findings suggest that hyperthyroidism increases oxidative stress. Treatment with thyroxin produces oxidative stress, which is reversed in hypothyroidism. Hence, it is contemplated that there exist some relationship between the dynamics of oxidative stress and thyroid state

 

KEY WORDS       Free Radicals, Oxidative Stress, Antithyroid, and Thyroid gland.

 


INTRODUCTION:

Oxidative stress is a condition in which generation of free radicals and other reactive oxygen species overwhelms the endogenous antioxidant defense of the body. This oxidative stress has been implicated in a variety of pathological conditions such as diabetes mellitus, inflammation, cancer, aging, ischemia, atherosclerosis, liver damage etc.1 

 

Thyroid hormones play a crucial role in the regulation of mitochondrial oxidative metabolism.2  High concentrations of thyroid hormones may change the metabolism of oxygen in the cells and stimulate the production of free radicals.3  It was observed that in course of hyperthyroidism, oxidative stress and the peroxidation of lipids can be generated.4  Acceleration of the basal metabolic rate and the energy metabolism of tissues in several mammalian species represent one of the major functions of thyroid hormones.5 Accumulating evidence has suggested that the hypermetabolic state in hyperthyroidism is associated with increase in free radical production and lipid peroxide level6,7, whereas the hypometabolic state induced by hypothyroidism is associated with a decrease in free radical production8 and in lipid peroxidation products.9 The changes in the levels of the scavengers α-tocopherol10, glutathione 11, 12 and coenzyme Q 10, and activities of antioxidant enzymes7 in various tissues were found to be imbalanced and often

 

opposite.  It is worthwhile to mention that some of the  antithyroid drugs have antioxidant effects13, 14. It was observed that both methimazole and propylthiouracil abolished or reduced the oxygen radical production by complement-attacked thyroid cells and decreased cytokine production3.

 

The roles of thyroid hormones in metabolic pathways are well known however, their involvement in lipid peroxidation and antioxidant enzyme activities is not known. The thyroid hormones may play a crucial role in inducing the generation of generalized oxidative stress. Variations of the levels of thyroid hormones can be one of the main physiological modulators of in vivo cellular oxidative stress due to their known effects on mitochondrial respiration15.

 

In the present work, we evaluated thyroid function tests and oxidative stress parameters in different thyroid state i.e. hypothyroidism and hyperthyroidism. We aimed at examining the possible relationship between the dynamics of oxidative stress and thyroid state.

EXPERIMENTAL:     

Animals:

Healthy male Sprague-Dawley species rats weighing between 180-230 g. were housed in a clean polypropylene cage under the controlled conditions of temperature (25 ± 20C), humidity (55 ± 2%) and light (dark/light -12/12hr cycle). They received standard rodent pellet food (Lipton India) and water ad libitum. All the rats were kept under the same experimental conditions. The experimental protocol was approved by Institutional Animal Ethical Committee (IAEC) constituted for the purpose of control and supervision of experimental animals by Ministry of Environment & Forest, Government of India, New Delhi.

 

Chemicals:

Trichloroacetic acid, Thiobarbituric acid and 5,5 dithio-bis-2 nitro benzoic acid (DTNB) from Sigma Aldrich Co. U.S.A. Metaphosphoric acid, pyrogallol, hydrogen peroxide and methimazole were purchased from LOBA Chemie. All other chemicals were of analytical grades.

 

Method:

Animals were randomly divided into three groups: control, hypothyroid, and hyperthyroid. Hypothyroidism was induced by administering methimazole in drinking water (0·02% w/v) for 15 days. Hyperthyroidism was induced by daily i.p. administration of tri-iodothyronine (T3) (100 µg/kg body weight) for 10 days in methimazole treated rats to induce mild hyperthyroidism in hypothyroid rats.

 

At the end of the protocol, blood was withdrawn from retro-orbital plexus. The parameters like lipid peroxidation and endogenous antioxidant enzymes, superoxide dismutase, catalase were determined in erythrocyte lysate while reduced glutathione was determined in blood to find out the extent of oxidative stress.  Same blood was utilized for testing of thyroid function by determining the levels of T3 T4 & TSH.

 

ASSESSMENT OF OXIDATIVE STRESS:

 

Lipid Peroxidation Assay: 16

For determination of lipid peroxidation (LPO,) the blood was withdrawn from retro-orbital plexus and was taken in the centrifuge tube containing anticoagulant. From this 5% suspension of RBC in 0.1M phosphate buffer saline was prepared. To the 2ml of this 5% suspension, 2ml of 28% trichloroacetic acid was added and centrifuged. After centrifugation the supernatant was separated. To the 4ml of supernatant 1ml of 1% thiobarbituric acid was added, heated in boiling water for 60 minute and cooled immediately. The absorbance was measured spectrophotometrically at 532 nm. The lipid peroxidation was calculated on the basis of the molar extinction coefficient of malondialdehyde (1.56 ´ 105) and expressed in terms of nanomoles of MDA/g Hb.

 

Reduced glutathione Assay: 17

Glutathione activity was measured in whole blood. 0.2ml of whole blood was added to 1.8 ml of distilled water followed by 3.0ml of precipitating mixture (1.67 g. of metaphosphoric acid, 0.2gms of EDTA, 30 g. NaCl to make 100ml of solution). It was centrifuged at 2000 rpm for 5 minutes. 1ml. supernatant was added to 1.5ml of phosphate solution followed by addition of 0.5ml of DTNB reagent. The optical density was measured at 412nm using spectrophotometer.

Super Oxide Dismutase (SOD) Assay: 18

The activity of superoxide dismutase enzyme (SOD) was determined in the erythrocyte lysate prepared from the 5% RBC suspension. To 50ml of the lysate, 2ml of 75mM of tris HCl buffer (pH 8.2), 0.6 ml of 30mM of EDTA and 0.3ml of 2mM of pyrogallol were added. An increase in the absorbance was measured at 420nm for 3 minutes using spectrophotometer. One unit of enzyme activity is 50% inhibition of the rate of auto-oxidation of pyrogallol, as determined by change in absorbance/minute at 420nm.

 

Catalase Assay: 19

The activity of catalase enzyme was determined in erythrocyte lysate. 50ml of the lysate was taken and added to a test tube containing 2ml of phosphate buffer (pH 7.0) and then 1ml of 30mM of H2O2 was added to it. The decrease in absorbance was measured at 240 nm for 1 minutes using spectrophotometer.

 

Statistical Analysis:

The resulted values were expressed as mean ± SEM (n=6). All data were analysed by One–Way ANOVA followed by Newman-Keuls multiple comparison test. The level of significance was considered at P<0.05.

RESULTS:

In the present investigation it was observed that the levels of T3 and T4 were decreased in the plasma of hypothyroidised rats when compared to T3 and T4 levels of normal control rats. Whereas the level of TSH was increased significantly as compared to normal control rats. While in case of hyperthyroidised rats the levels of T3 and T4 were found increased significantly whereas level of TSH was decreased significantly. (Table no. 1)

Table 1 : Effect of thyroid state on thyroid function tests in rats

Groups

T3

T4

TSH

Control

35.46 ± 0.9240

2.76 ± 0.09281

1.802 ± 0.04969

Hypothyroid

32.33 ± 0.5970#

1.283 ± 0.1146*

12.55 ± 0.4333*

Hyperthyroid

56.93 ± 0. 1.45*

6.377 ± 0.1110*

0.8633 ± 0.03703

Values are mean ± SEM(n=6). *P<0.001, #P<0.05 and **P<0.01, when compared to respective controls.

 

Further, there was significant increase in the parameters of oxidative stress such as levels of lipid peroxidation (Fig. 1) and reduced glutathione (Fig. 2) in case of hyperthyroidism group as compared to normal control rats.  Furthermore, the increase in lipid peroxidation was found to be attenuated in the rats with hypothyroidism. While, it was observed that the levels of the another oxidative stress markers i.e. SOD (Fig. 3) and Catalase (Fig. 4) were significantly decreased in hyperthyroidism group as compared to normal control rats. The levels of these markers in hypothyroidised rats were more or less as that of vehicle treated control group.


Table 2 : Effect of thyroid state on various parameters of oxidative stress in rats

Groups

Lipid peroxidation (nm MDA/g Hb)

Superoxide dismutase (units/mg protein)

Catalase (units/mg protein)

Reduced glutathione       (mm DTNB conjugated/g Hb)

Control

2.09± 0.0587

32.68± 1.128

279.5± 9.265

12.12±0.2800

Hypothyroid

1.957±0.0738

35.31±2.357

317.1± 13.77

12.58±0.3156

Hyperthyroid

4.282±0.3845*

26.15±0.562#

234.9± 7.08**

11.10±0.275#

Values are mean ± SEM(n=6). *P<0.001and #P<0.05 when compared to respective controls.


DISCUSSION:

Reduction in the metabolic reactions occurring along with hypothyroidism also cause lowered lipid peroxidation level and decreased generation of free radicals8. Propylthiouracil has been reported to be as an anti-thyroid drug with noticeable in-vivo as well as in-vitro inhibitory effect on H2O2 production in neutrophils. Most of the researchers have been reported the effects of thyroid hormones on lipid peroxidation but the results are rather contradictory.

 

Fig. 1. The effect of thyroid state on lipid peroxidation (LPO)

Values are mean ± SEM (n=6). *P<0.001 when compared to control.

 

A considerable body of evidence now exists attesting the involvement of uncontrolled oxidative activity as a general mechanism of tissue damage in a variety of pathological conditions 20. In particular, it has been suggested that the increase in reactive oxygen species induced by thyroid hormone leads to an oxidative stress condition in liver21 and in cardiac and some skeletal muscles2 with a consequent lipid peroxidative response. Although the pathophysiological consequences of the

 

Fig. 2. The effect of thyroid state on reduced glutathione (GSH)

 

Values are mean ± SEM (n=6). *P<0.05 when compared to control.

 

 

accelerated lipid peroxidation are not yet fully elucidated, this biochemical change is thought to be responsible for some complications of hyperthyroidism.

 

We found suppressed TSH level, with elevation of T4 and T3, increased lipid peroxidation and decreased superoxide dismutase (SOD), catalase and glutathione in hyperthyrodism as observed in oxidative stress. Methimazole effectively reduced the lipid peroxidation. and the elevated levels of thyroid hormones. Also, we found that superoxide dismutase (SOD), catalase and glutathione (GSH) values higher in hypothyroid rats, in comparison to the control rats. In conclusion, there was increase in oxidative stress in hyperthyroidism and the normalization by the treatment with methimazole exhibited the protective effect against oxidative stress.

 

 

Fig. 3. The effect of thyroid state on superoxide dismutase (SOD)

Values are mean ± SEM (n=6). *P<0.05 when compared to control.

 


One of the study indicated that hypothyroidism reduces oxidative damage in cerebral, hepatic and cardiac tissues of rats; however, administration of high dose of thyroxin in addition to induced hypothyroidism increases oxidative damage in the same tissues and this damage can not be prevented despite the increase in antioxidant system activity22.

 

Reports as well as our findings suggested that hyperthyroidism increases oxidative stress. Treatment with thyroxin produces oxidative stress, which is reversed in hypothyroidism. Hence, it is contemplated that there exist some relationship between the dynamics of oxidative stress and thyroid state.

Fig. 4. The effect of thyroid state on catalase (CAT)

Values are mean ± SEM (n=6). *P<0.001 when compared to control.

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Received on 24.03.2008          Modified on 30.03.2008

Accepted on 08.04.2008          © RJPT All right reserved

Research J. Pharm. and Tech. 1(1): Jan.-Mar. 2008; Page 14-17