# The Interaction of Oxidative Stress Response with Cytokines in the Thyrotoxic Rat: Is There a Link?

Authors: Balahan Makay; Ozer Makay; Cigdem Yenisey; Gokhan Icoz; Gokhan Ozgen; Erbil Unsal; Mahir Akyildiz; Enis Yetkin
Journal: Mediators of Inflammation
 (2009)

---

## Abstract

Oxidative stress is regarded as a pathogenic factor in hyperthyroidism. Our purpose was to determine the relationship between the oxidative stress and the inflammatory cytokines and to investigate how melatonin affects oxidative damage and cytokine response in thyrotoxic rats. Twenty-one rats were divided into three groups. Group A served as negative controls. Group B had untreated thyrotoxicosis, and Group C received melatonin. Serum malondialdehyde (MDA), glutathione (GSH), glutathione reductase (GR), glutathione peroxidase (GPx), and nitric oxide derivates (NO•x), and plasma IL-6, IL-10, and TNF-alpha were measured. MDA, GSH, NO•x, IL-10, and TNF-alpha levels increased after L-thyroxine induction. An inhibition of triiodothyronine and thyroxine was detected, as a result of melatonin administration. MDA, GSH, and NO•x levels were also affected by melatonin. Lowest TNF-alpha levels were observed in Group C. This study demonstrates that oxidative stress is related to cytokine response in the thyrotoxic rat. Melatonin treatment suppresses the hyperthyroidism-induced oxidative damage as well as TNF-alpha response.


---

## Body

## 1. Introduction

Hyperthyroidism is associated with an increased
metabolic rate due to increments in the rate of oxygen consumption in target
tissues [1]. Acceleration of aerobic metabolism by thyroid hormones enhances
the generation of oxidative stress [2]. Oxidative stress is regarded as a
pathogenic factor in hyperthyroidism. Clinical and experimental studies
reported increased levels of free oxygen radicals and a decreased antioxidant
status in thyrotoxicosis [3–6].Free oxygen radicals are general mediators
of signal transduction pathways, which are able to induce cytokine production
from various cell types [7–10]. Studies
attesting to the role of antioxidants, of which potentially inhibit the
activation of oxidant-mediated transcription factors, reported that these
antioxidants also prevent the transcriptional activation of inflammatory
cytokines [11–13]. These
studies suggest that antioxidants may play a role in decreasing the immune
response by suppressing the oxidative stress. Since these findings may be of
clinical relevance in the thyrotoxic patient, herein we aimed to investigate (1)
whether oxidative stress affects the production of cytokines IL-6, IL-10, and
TNF-alpha and (2) whether these alterations are inhibited by melatonin-based
antioxidative therapy.


## 2. Methods

Twenty-one
12-week-old male Wistar albino rats with a body weight of 250–300 g were
included in the study. All were housed in groups of 7 in identical wire-bottomed
cages with a 12 hour day-night cycle, at a constant room temperature of24±1°C.
The animals were acclimatized to these conditions for 10 days before the
experiment. Standard rat food and water was freely available. All rats were
treated according to
the UFAW Handbook on the Care and Management of Laboratory Animals (Blackwell,
7th ed, Blackwell Science, Iowa, 1999) and Principles of Laboratory Animal Care
(NIH publication no. 86-23, revised 1985). The protocol was approved by Ege
University Ethics Committee of Experimental Studies and Ege University
,
Department of Experimental Surgery.Rats were randomized to the following
groups according to the treatment to which each was subjected and were
sacrificed at the end of the 3-weeks treatment:(1)
Group A (n=7), the
sham group, was not subjected to any procedure, except receiving daily
intraperitoneal injections of 0.9% saline solution;(2)
Group B (n=7), the
untreated (control) group, underwent intraperitoneal L-thyroxine administration
for induction of hyperthyroidism, for 21 days and at a daily dose of 0.2 mg/kg. L-thyroxine was dissolved using 0.01 N NaOH, and the final solution
was prepared with 0.9% saline solution;(3)
Group C (n=7), the
treatment group, underwent L-thyroxine as in Group B and besides, received
daily (each 24 hours) 3 mg/kg melatonin for 21 days (Ilsan Iltas Laboratories,
Istanbul), intraperitoneally.


### 2.1. Laboratory Measurements

All evaluations were done by a single biochemist
blinded to the study. Blood was collected through
heart puncture to estimate thyroid function tests (TSH, FT3, and FT4),
oxidative stress markers (malondialdehyde [MDA], glutathione [GSH], glutathione
reductase [GR], glutathione peroxidase [GPx], and nitric oxide [NO•]), and
cytokines (IL-6, IL-10 and, TNF-alpha). Blood samples were centrifuged at 2500 g for 5 minutes at room
temperature. The serum was removed and stored at −80°C for later studies. Free
T3 (triiodothyronine) (FT3), free T4 (thyroxine) (FT4), and TSH (thyroid stimulating hormone) analyses
were carried out with a chemiluminescent enzyme
radioimmunometric assay by using the IMMULATE 2000 automated hormone analyzer
(Diagnostic Products, Los Angeles, Calif, USA). The
reproducibility of the methods, established before the study by conducting 2
additional independent experiments, was good with clear normative limits and a
95% agreement.


### 2.2. Measurement of MDA

Lipid peroxidation (LPO) is frequently investigated
in biomedical research, and the assays for thiobarbituric acid-reactive
substances (TBARSs)
are more widely used than any other index of LPO in biological samples.
Thiobarbituric acid reacts with LPO aldehydes, such as malondialdehyde (MDA).
Therefore, assessment of TBARS is a useful index of oxidative deterioration and LPO
determination in body fluids. MDA levels were determined spectrophotometrically at 532 nm by the
method of Ohkowa et al. [14]. MDA formed a colored complex in the presence of
thiobarbituric acid, which was detectable by measurement of absorbance at 532 nm. Absorbance was measured with Shimadzu UV-160 spectrophotometer. 1,1′,3,3′ tetraethoxypropane
was used as a standard. Levels were calculated as nmol/mL.


### 2.3. Measurement of GSH

Total glutathione content was measured according to
the method of Tietze [15]. In brief, 0.5 mL sample or standard solution was
mixed with 0.25 mL of 1 mol/L sodium phosphate buffer (pH 6.8) and 0.5 mL
5-5′-dithiobis-(2-nitrobenzoic acid) (DTNB, 0.8 g/L in the phosphate buffer)
for 5 minutes. Then, the absorbance was measured at 412 nm using a Shimadzu
UV-160 spectrophotometer. The GSH concentration was determined using standard
aqueous solutions of GSH. Results were expressed as mg/dL.


### 2.4. Measurement of GR

Glutathione reductase was assayed by following the
oxidation of NADPH at 340 nm at37°C [16]. The reaction was initiated by the addition of
50 μL supernatant to 1 mL of assay mixture containing 50 mmol/L Tris, pH 7.6,
100 μmol/L Na2EDTA, 4 mmol/L GSSG, and 120 μmol/L NADPH. A blank
cuvet was prepared in which the sample was replaced with water. The reaction
was linear for 2 to 3 minutes. Hemoglobin in erythrocyte lysates was estimated
using the method of Drabkin and Austin [17]. Activity in samples was normalized
for hemoglobin content and is expressed in U/g Hb.


### 2.5. Measurement of GPx

Glutathione peroxidase activity was determined by a
minor modification of the method of Paglia and Valentine [18]. A 50 μL
supernatant was transferred to a 1 mL quartz cuvet, containing 950 μL of the
reaction mixture (Tris buffer, 50 mmol/L, pH 7.6, containing per liter, 1 mmol
of Na2EDTA, 2 mmol of reduced glutathione, 0.2 mmol NADPH, 4 mmol
sodium azide, and 1000 U of glutathione reductase). The mixture was incubated 5
minutes in 37°C. Then, the reaction
was initiated by adding 25 μL of H2O2, 8.8 mmol/L (% 30).
The decrease in NADPH absorbance at 340 nm was followed for 3 minutes. The
nonenzymic reaction rate (blank) was determined by substituting water for the
supernatant. The decrease in NADPH absorbance was recorded. Hemoglobin was
evaluated as described above. Activity in samples was normalized for hemoglobin
content and is expressed in U/g Hb.


### 2.6. Measurement of NO• Derivative Content

NO•x (nitrite
+ nitrate) was assayed by a modification of cadmium-reduction method as mentioned
by Navarro-Gonzàlves [19]. The nitrite produced was determined by diazotization
of sulphanilamide and coupling to naphthlethylene diamine. For the measurement
of NO•x, a 400 μL sample was denatured by adding 80 μL 30% ZnSO4 solution, stirring and then centrifuging at 10 000 xg for 20 minutes at 4°C. First, we activated Cd granules using CuSO4 solution in glycine-NaOH buffer. Then, 100 μL of deproteinized samples and standards were
added. This reaction, using pretreatment of samples to reduce nitrate to
nitrite, can be accomplished by catalytic reactions using enzyme or Cd. The
samples were analyzed spectrophotometrically using a microplate reader and
quantified automatically against KNO3 standard curve. Results were
expressed as μM/L.


### 2.7. Measurement of IL-6

Plasma IL-6 levels were determined via a commercial
rat ELISA kit (IBL Co., Ltd. Hamburg, Germany),
catalogue and lot number 17196. The test results were calculated by bioelisa
reader ELx800 using standard
curve.  The kit measurement range was
11.72–750 pg/mL.
Interassay values were calculated for three different measurement values (34.83,
71.71, and 293.31 pg/ml), and CV values were found to be 2.3%, 1.9%, and 1.7%,
respectively, (n=10). Besides, intra-assay values determined three different
measurement values (33.57, 70.45, and 293.45 pg/mL), and CV values were found
to be 8.8%, 7.7%, and 4.9%, respectively, (n=10). We did not use haemolysed
samples.


### 2.8. Measurement of IL-10

IL-10 levels were determined via a commercial human ELISA kit
(IMMUNOTECH; Beckman Coulter Company, Marseille Cedex, France). Test results
were calculated by bioelisa reader ELx800 using standard curve. The kit measurement range was 0–2000 pg/ml. Kits’
performance characteristics were performed. Intra-assay precision was
determined by assaying sera 10 times, and CVs ranged between 3.3 and 4.0%.
Interassay precision was determined by assaying samples 10 times in independent
assays. CVs ranged between 5.6 and 8.6%.
We did not use haemolysed samples.


### 2.9. Measurement of TNF-Alpha

TNF-α levels were determined
via a commercial rat ELISA kit (IBL Co., Ltd. Hamburg ,
Germany), catalogue number 17194. Test results were calculated by bioelisa reader
ELx800 using standard
curve. The kit measurement range was 0.39–25 ng/mL. Kits’
performance characteristics were performed. Interassay values were calculated
for three different measurement values (0.36, 1.69, and 7.03 ng/mL), and CV values
were found to be 8.8%, 3.0%, and 1.0%, respectively, (n=10). Besides,
intra-assay values determined three different measurement values (0.33, 1.69,
and 7.03 ng/mL), and CV values were found to be 8.8%, 3.0%, and 1.0%,
respectively, (n=10). Besides, intra-assay values determined three different
measurement values (0.33, 1.68, and 6.7 ng/mL), and CV values were found to be 9.1,
9.5, and 4.3, respectively, (n=10). We did not use haemolysed samples.


### 2.10. Statistical Analysis

All analyses were
performed by using SPSS Statistical Analysis software (release 10.0, Chicago, Ill, USA). All data are
expressed as mean ± standard deviation. To search difference between the groups,
Kruskal-Wallis one-way analysis was used. Depending on the homogeneity of the
variances, Mann-WhitneyU-test and Bonferonni post hoc test were used in pair
comparison. Correlations between oxidative stress
parameters and serum concentrations of cytokines were studied by regression
analysis. P-values of less than .05 were considered to be significant.


## 2.1. Laboratory Measurements

All evaluations were done by a single biochemist
blinded to the study. Blood was collected through
heart puncture to estimate thyroid function tests (TSH, FT3, and FT4),
oxidative stress markers (malondialdehyde [MDA], glutathione [GSH], glutathione
reductase [GR], glutathione peroxidase [GPx], and nitric oxide [NO•]), and
cytokines (IL-6, IL-10 and, TNF-alpha). Blood samples were centrifuged at 2500 g for 5 minutes at room
temperature. The serum was removed and stored at −80°C for later studies. Free
T3 (triiodothyronine) (FT3), free T4 (thyroxine) (FT4), and TSH (thyroid stimulating hormone) analyses
were carried out with a chemiluminescent enzyme
radioimmunometric assay by using the IMMULATE 2000 automated hormone analyzer
(Diagnostic Products, Los Angeles, Calif, USA). The
reproducibility of the methods, established before the study by conducting 2
additional independent experiments, was good with clear normative limits and a
95% agreement.


## 2.2. Measurement of MDA

Lipid peroxidation (LPO) is frequently investigated
in biomedical research, and the assays for thiobarbituric acid-reactive
substances (TBARSs)
are more widely used than any other index of LPO in biological samples.
Thiobarbituric acid reacts with LPO aldehydes, such as malondialdehyde (MDA).
Therefore, assessment of TBARS is a useful index of oxidative deterioration and LPO
determination in body fluids. MDA levels were determined spectrophotometrically at 532 nm by the
method of Ohkowa et al. [14]. MDA formed a colored complex in the presence of
thiobarbituric acid, which was detectable by measurement of absorbance at 532 nm. Absorbance was measured with Shimadzu UV-160 spectrophotometer. 1,1′,3,3′ tetraethoxypropane
was used as a standard. Levels were calculated as nmol/mL.


## 2.3. Measurement of GSH

Total glutathione content was measured according to
the method of Tietze [15]. In brief, 0.5 mL sample or standard solution was
mixed with 0.25 mL of 1 mol/L sodium phosphate buffer (pH 6.8) and 0.5 mL
5-5′-dithiobis-(2-nitrobenzoic acid) (DTNB, 0.8 g/L in the phosphate buffer)
for 5 minutes. Then, the absorbance was measured at 412 nm using a Shimadzu
UV-160 spectrophotometer. The GSH concentration was determined using standard
aqueous solutions of GSH. Results were expressed as mg/dL.


## 2.4. Measurement of GR

Glutathione reductase was assayed by following the
oxidation of NADPH at 340 nm at37°C [16]. The reaction was initiated by the addition of
50 μL supernatant to 1 mL of assay mixture containing 50 mmol/L Tris, pH 7.6,
100 μmol/L Na2EDTA, 4 mmol/L GSSG, and 120 μmol/L NADPH. A blank
cuvet was prepared in which the sample was replaced with water. The reaction
was linear for 2 to 3 minutes. Hemoglobin in erythrocyte lysates was estimated
using the method of Drabkin and Austin [17]. Activity in samples was normalized
for hemoglobin content and is expressed in U/g Hb.


## 2.5. Measurement of GPx

Glutathione peroxidase activity was determined by a
minor modification of the method of Paglia and Valentine [18]. A 50 μL
supernatant was transferred to a 1 mL quartz cuvet, containing 950 μL of the
reaction mixture (Tris buffer, 50 mmol/L, pH 7.6, containing per liter, 1 mmol
of Na2EDTA, 2 mmol of reduced glutathione, 0.2 mmol NADPH, 4 mmol
sodium azide, and 1000 U of glutathione reductase). The mixture was incubated 5
minutes in 37°C. Then, the reaction
was initiated by adding 25 μL of H2O2, 8.8 mmol/L (% 30).
The decrease in NADPH absorbance at 340 nm was followed for 3 minutes. The
nonenzymic reaction rate (blank) was determined by substituting water for the
supernatant. The decrease in NADPH absorbance was recorded. Hemoglobin was
evaluated as described above. Activity in samples was normalized for hemoglobin
content and is expressed in U/g Hb.


## 2.6. Measurement of NO• Derivative Content

NO•x (nitrite
+ nitrate) was assayed by a modification of cadmium-reduction method as mentioned
by Navarro-Gonzàlves [19]. The nitrite produced was determined by diazotization
of sulphanilamide and coupling to naphthlethylene diamine. For the measurement
of NO•x, a 400 μL sample was denatured by adding 80 μL 30% ZnSO4 solution, stirring and then centrifuging at 10 000 xg for 20 minutes at 4°C. First, we activated Cd granules using CuSO4 solution in glycine-NaOH buffer. Then, 100 μL of deproteinized samples and standards were
added. This reaction, using pretreatment of samples to reduce nitrate to
nitrite, can be accomplished by catalytic reactions using enzyme or Cd. The
samples were analyzed spectrophotometrically using a microplate reader and
quantified automatically against KNO3 standard curve. Results were
expressed as μM/L.


## 2.7. Measurement of IL-6

Plasma IL-6 levels were determined via a commercial
rat ELISA kit (IBL Co., Ltd. Hamburg, Germany),
catalogue and lot number 17196. The test results were calculated by bioelisa
reader ELx800 using standard
curve.  The kit measurement range was
11.72–750 pg/mL.
Interassay values were calculated for three different measurement values (34.83,
71.71, and 293.31 pg/ml), and CV values were found to be 2.3%, 1.9%, and 1.7%,
respectively, (n=10). Besides, intra-assay values determined three different
measurement values (33.57, 70.45, and 293.45 pg/mL), and CV values were found
to be 8.8%, 7.7%, and 4.9%, respectively, (n=10). We did not use haemolysed
samples.


## 2.8. Measurement of IL-10

IL-10 levels were determined via a commercial human ELISA kit
(IMMUNOTECH; Beckman Coulter Company, Marseille Cedex, France). Test results
were calculated by bioelisa reader ELx800 using standard curve. The kit measurement range was 0–2000 pg/ml. Kits’
performance characteristics were performed. Intra-assay precision was
determined by assaying sera 10 times, and CVs ranged between 3.3 and 4.0%.
Interassay precision was determined by assaying samples 10 times in independent
assays. CVs ranged between 5.6 and 8.6%.
We did not use haemolysed samples.


## 2.9. Measurement of TNF-Alpha

TNF-α levels were determined
via a commercial rat ELISA kit (IBL Co., Ltd. Hamburg ,
Germany), catalogue number 17194. Test results were calculated by bioelisa reader
ELx800 using standard
curve. The kit measurement range was 0.39–25 ng/mL. Kits’
performance characteristics were performed. Interassay values were calculated
for three different measurement values (0.36, 1.69, and 7.03 ng/mL), and CV values
were found to be 8.8%, 3.0%, and 1.0%, respectively, (n=10). Besides,
intra-assay values determined three different measurement values (0.33, 1.69,
and 7.03 ng/mL), and CV values were found to be 8.8%, 3.0%, and 1.0%,
respectively, (n=10). Besides, intra-assay values determined three different
measurement values (0.33, 1.68, and 6.7 ng/mL), and CV values were found to be 9.1,
9.5, and 4.3, respectively, (n=10). We did not use haemolysed samples.


## 2.10. Statistical Analysis

All analyses were
performed by using SPSS Statistical Analysis software (release 10.0, Chicago, Ill, USA). All data are
expressed as mean ± standard deviation. To search difference between the groups,
Kruskal-Wallis one-way analysis was used. Depending on the homogeneity of the
variances, Mann-WhitneyU-test and Bonferonni post hoc test were used in pair
comparison. Correlations between oxidative stress
parameters and serum concentrations of cytokines were studied by regression
analysis. P-values of less than .05 were considered to be significant.


## 3. Results

### 3.1. Thyroid State

The thyroid state of different groups of animals was
characterized by the data reported in Table1. The values indicate significant
rises in FT3 and FT4 levels after L-thyroxine
administration (P<.01). It was also found that melatonin treatment
significantly decreased the concentrations of FT3 and FT4 when compared to rats with hyperthyroidism in the untreated group (P<.01).Table 1
Comparison of thyroid function tests.
nGroup AGroup BGroup CFT3
(pg/mL)71.49±0.268.68±3.69*3.28±2.98**FT4 (ng/dL)71.44±0.444.62±2.18*1.94±1.22**TSH (μIU/mL)70.25±0.020.15±0.010.20±0.36Values were
expressed as mean ± standard
deviation.P*<.01;
significant when compared with Group A.P**<.01;
significant when compared with Group B.


### 3.2. Oxidative Stress and Response to Oxidative Stress

MDA, GSH, GR, GPx, and NO•x levels of
control and treatment groups are presented in Table 2. Comparison among the
groups revealed that MDA, GSH, and NO•x levels in
hyperthyroid rats were significantly higher than the negative control and
treatment groups (P<.01).Table 2
Comparison
of MDA, GSH, GR, GPx, and NO• derivate
levels.
GroupnMDA
(μmol/L)GSH
(mg/dL)GR (U/g Hb)GPx
(U/g Hb)NO
(μM/L)A73.42±1.470.26±0.060.83±0.344.02±1.516.72±1.55B74.80±1.23*0.43±0.06*1.05±0.393.48±0.7666.0±58.54*C72.31±0.71**0.35±0.07**0.78±0.313.88±1.1747.11±15.18**Values were
expressed as mean ± standard
deviation.P*<.01;
significant when compared with Group A.P**<.01;
significant when compared with Group B.


## 3.1. Thyroid State

The thyroid state of different groups of animals was
characterized by the data reported in Table1. The values indicate significant
rises in FT3 and FT4 levels after L-thyroxine
administration (P<.01). It was also found that melatonin treatment
significantly decreased the concentrations of FT3 and FT4 when compared to rats with hyperthyroidism in the untreated group (P<.01).Table 1
Comparison of thyroid function tests.
nGroup AGroup BGroup CFT3
(pg/mL)71.49±0.268.68±3.69*3.28±2.98**FT4 (ng/dL)71.44±0.444.62±2.18*1.94±1.22**TSH (μIU/mL)70.25±0.020.15±0.010.20±0.36Values were
expressed as mean ± standard
deviation.P*<.01;
significant when compared with Group A.P**<.01;
significant when compared with Group B.


## 3.2. Oxidative Stress and Response to Oxidative Stress

MDA, GSH, GR, GPx, and NO•x levels of
control and treatment groups are presented in Table 2. Comparison among the
groups revealed that MDA, GSH, and NO•x levels in
hyperthyroid rats were significantly higher than the negative control and
treatment groups (P<.01).Table 2
Comparison
of MDA, GSH, GR, GPx, and NO• derivate
levels.
GroupnMDA
(μmol/L)GSH
(mg/dL)GR (U/g Hb)GPx
(U/g Hb)NO
(μM/L)A73.42±1.470.26±0.060.83±0.344.02±1.516.72±1.55B74.80±1.23*0.43±0.06*1.05±0.393.48±0.7666.0±58.54*C72.31±0.71**0.35±0.07**0.78±0.313.88±1.1747.11±15.18**Values were
expressed as mean ± standard
deviation.P*<.01;
significant when compared with Group A.P**<.01;
significant when compared with Group B.


## 3.3. Plasma Cytokine Concentration

Levels of both IL-10 and TNF-alpha were significantly
enhanced after L-thyroxine administration (P<.01), while the rise in
IL-6 was insignificant (P<.05). Melatonin treatment was associated
with significant decreased TNF-alpha levels (P<.01) (Table 3). There
was a significant correlation between IL-10 concentration and both oxidative
stress parameters MDA and NO (r=0.48, P<.05 and r=0.39, P<.01,
resp.). A significant correlation also existed between serum TNF-alpha and MDA
(r=0.49, P<.01) as well as TNF-alpha and NO level (r=0.69, P<.01).
No correlations were found between TNF-alpha and thyroid hormone levels (P>.05).Table 3
Comparison
of plasma cytokine levels.
GroupnIL-6
(pg/mL)IL-10
(pg/mL)TNF-alpha
(ng/mL)A7324.66±26.2833.53±10.030.76±0.04B7345.99±109.2051.99±17.43*0.88±0.1*C7333.33±38.9256.00±10.330.75±0.05**Values were
expressed as mean ± standard
deviation.P*<.01;
significant when compared with Group A.P**<.01;
significant when compared with Group B.


## 4. Discussion

The findings of the study suggest that (1) thyrotoxicosis stimulates the oxidative stress response and the production of inflammatory cytokines (IL-10 and TNF-alpha), (2) there is a relationship between oxidative stress parameters and inflammatory cytokines, (3) melatonin prevents the increase in FT3 and FT4 caused by chronic T4 injection, and (4) melatonin-based antioxidative treatment inhibits thyroid hormone induced increases in MDA, GSH, NO•, and TNF-alpha levels. Meanwhile, to our knowledge, the latter observation, that melatonin acts on TNF-alpha levels, seems novel. We hypothesized that reactive oxygen intermediates, that were induced by thyrotoxicosis, act as a signal for the release of cytokines like IL-6, IL-10, and TNF-alpha. We documented the increased oxidative stress and antioxidant system capacity, together with response to allopurinol, in different time intervals, in another paper, which is soon going to be published elsewhere.Previous studies have provided evidence that free radicals increase during hyperthyroidism [3–6]. Whatever the pathways are, it is clear that T3-induced liver free-radical generation occurs in concomitance with enhanced respiratory burst activity and chemiluminescent response in both experimental and human studies [20, 21]. The evidence that oxidative stress occurs related to thyroid hormone induction is supported in the current study, since we have demonstrated significant increases in oxidative stress parameters like MDA and NO•x. Although some modalities have been suggested to prevent the development of oxidative stress during thyrotoxicosis, no management has yielded promising uniform success, clinically [22–24].Oxidative stress is paralleled by impaired antioxidant mechanisms; glutathione is one of these important systems [25]. In one of the few studies related to GSH in hyperthyroidism, GSH was stimulated in an experimental model [26]. In these studies, it was concluded that free radicals appear to stimulate GSH production in prolonged oxidative stress. This explanation seems plausible, since biochemical data of our study showed an increase in GSH.Several previous reports have tried to address the
contribution of thyrotoxicosis to the rise in inflammatory cytokines such as IL-1,
IL-6, IL-10, and TNF-alpha [13, 27–34]. Although
controversial results, almost all these studies suggest that cytokines may play
important roles in the process of hyperthyroidism. The only report
investigating the effect of TNF-alpha levels on thyroid hormone kinetics is the
one by Ongphiphadhanakul et al. These investigators found that TNF-alpha might
inhibit the peripheral generation of T3 from T4 and act
as an autocrine factor by decreasing thyroidal generation and release of T3 [33]. Our data is not in accordance with this finding, since no
correlation was found between TNF-alpha levels and thyroid hormones. In the present study, we demonstrated a significant IL-10
and TNF-alpha release after thyroid hormone induction, while the increase of
IL-6 was insignificant. Thus, our results do not support any role for IL-6 in the pathogenesis of
thyrotoxicosis.IL-6
acts as a systemic hormone [35] that may mediate the well-documented inhibitory effect of IL-1 on thyroid
cell functions [36]. In regard
to IL-6, our findings are in agreement with the data presented by Senturk et
al. [29] and Burggraaf et al. [32], since we could not present a statistically
significant difference between thyrotoxic rats and control subjects. We suggest
that this may be attributed to the duration of thyroid hormone induction or the
consequence that since IL-6 can be bounded to plasma proteins, which may
camouflage IL-6 immunoreactivity in serum, as previously suggested [29].In
contrast to IL-6, IL-10 is one of the most potent anti-inflammatory cytokines [37].
IL-10 interferes with cellular immunity in several ways and is known to
attenuate the expression and/or production of proinflammatory cytokines [38].
TNF-alpha is expressed as one of these cytokines [39]. Our data did not support
this, since both IL-10 and TNF-alpha levels were elevated after T4 induction. Interestingly, proinflammatory stimuli like TNF-alpha enhance its
secretion without any influence of IL-6 [40]. There is a lack of evidence
regarding IL-10 and nonautoimmune thyroid disorders. Data presented by de la
Vega et al. supported that Il-10 levels in multinodular glands did not reach a
significant difference versus normal subjects [41]. But the study population
they studied was only 15, where each group consisted of 5 patients.The link between free oxygen radicals and
inflammatory processes in hyperthyroidism was studied previously by Fernández
et al. [27]. They addressed that the effects of thyroid hormones on the
expression of nuclear factor kappa B (NF-κB) ligand are involved in the cytokine response seen
in hyperthyroidism. There is evidence that T3 administration to rats
show induced expression of NF-κB-responsive genes for TNF-alpha and IL-10 [13].
This was correlated with increases in the serum levels of the cytokines in the
same study.Even though deleterious effects of NO• in
oxidative processes [42], its production is accepted to be induced by
proinflammatory cytokines [43]. In our model, thyrotoxicosis resulted in
obvious increased NO•x levels,
and there was a correlation between NO•x and
cytokine levels. This correlation suggests a link between NO and cytokine
response in the thyrotoxic rat. This might simply mean that while a probable
contributory role for these cytokines is suggested, it seems not to be the only
factor involved in the complex pathogenesis in thyrotoxicity-related oxidative
stress.Melatonin, after discovered as a free radical
scavenger in 1993 [44], has been reported to be protective in various models of
oxidative stress, both through its free radical scavenging effect as well as by
directly increasing antioxidant activity [45]. Although melatonin has been
proposed to be useful for the prevention of oxidative stress during
hyperthyroidism [23, 46], to our knowledge, there has been no report studying
the effects of antioxidative therapy on serum concentrations of IL-6, IL-10,
and TNF-alpha in the thyrotoxic rat. The hypothesis that melatonin is protective
against free oxygen radicals was further supported by our observation that
after melatonin treatment, increased MDA, GSH, and NO•x levels
decreased significantly. Besides, thyroid hormone and TNF-alpha levels after
melatonin treatment were lower than in untreated rats, suggesting an inhibitory
effect of melatonin on T3, T4, and TNF-alpha. The
decrement in TNF-alpha levels did not relate with the decrease of thyroid
homone levels, suggesting a direct antioxidant effect of melatonin on TNF-alpha.
Studies on the hypothesis of an inhibitory effect of melatonin on thyroid
hormones documented contradictory results [47, 48]. In fact, melatonin is known
with its stimulating effect on antioxidative enzymes, such as GR and GPx [49].
These enzymes convert oxidized glutathione to GSH, leading to increased GSH
activity. However, in our study, melatonin administration did not result in
increased GR and GPx levels. Moreover, it resulted in decreased GSH. This
paradox might be attributed to differences in type and manner of melatonin
administration. Melatonin suggests an immunotherapeutic potential, since it inhibits
TNF-alpha. Whatever the mechanism is, the fact that melatonin counteracts the increase of TNF can
help toward an understanding of the complex nature of TNF induction in
hyperthyroidism.Collectively,
data presented in this work support a link between thyroid hormone-induced
oxidative stress and cytokine response. It provides new insights into the role
of IL-10 and TNF-alpha in thyrotoxicosis. Therapy for increased oxidative
stress with melatonin significantly reduces triiodothyronine, thyroxine, IL-10,
and TNF-alpha levels. The fact that circulating cytokines might differ from
those in different tissues is a limitation of this study. Although the expression
of cytokines seems to be involved with thyrotoxicosis-induced oxidative stress,
this relation remains to be studied in future qualitative studies.



---

*Source: 391682-2009-03-30.xml*