Investigation of zinc and copper levels in methimazole-induced hypothyroidism: relation with the oxidant-antioxidant status.
ABSTRACT Thyroid hormones are associated with the oxidative and antioxidative status of the organism. Depression of metabolism by hypothyroidism has been reported to decrease oxidant production and thus protect tissues against oxidant damage. The purpose of the present study was to investigate Zn and Cu levels in MMI-induced hypothyroidism and to show whether there is a connection between these trace elements and the oxidant-antioxidant status in experimental hypothyroidism. 3-Nitrotyrosine was measured as a marker of nitro-oxidative stress. In order to examine the antioxidant status of MMI-induced hypothyroidism in rats, GSH and SOD levels were determined as well. Significantly decreased 3-nitrotyrosine, Cu and Zn levels were observed in our experimental model when compared with the controls. On the other hand, GSH and SOD levels remained constant. It may be suggested that Cu and Zn serve as antioxidant molecules and exert their effects in an indirect manner to reduce oxidative stress in experimental hypothyroidism.
Article: Ameliorative effect of vitamin C on alterations in thyroid hormones concentrations induced by subchronic coadministration of chlorpyrifos and lead in wistar rats.[show abstract] [hide abstract]
ABSTRACT: The present study evaluated the ameliorative effect of vitamin C on alteration in thyroid hormones induced by low-dose subchronic coadministration of chlorpyrifos (CPF) and lead (Pb). Forty Wistar rats were divided into 4 groups of 10 animals each. Groups I and II were administered soya oil (2 mL/kg) and vitamin C (100 mg/kg), respectively. Group III was coadministered CPF (4.25 mg/kg ~1/20th LD(50)) and Pb (250 mg/kg ~1/20th LD(50)), respectively. Group IV was pretreated with vitamin C (100 mg/kg) and then coadministered with CPF (4.25 mg/kg) and Pb (250 mg/kg), 30 min later. The regimens were administered by gavage for a period of 9 weeks. The marginal decrease in serum triiodothyronine and thyroxine and the significant increase in the concentrations of thyroid stimulating hormone and malonaldehyde in the group coadministered with CPF and Pb were ameliorated by vitamin C partly due to its antioxidant properties.Journal of thyroid research. 01/2011; 2011:214924.
Investigation of Zinc and Copper Levels
in Methimazole-Induced Hypothyroidism:
Relation with the Oxidant-Antioxidant Status
(experimental hypothyroidism / oxidant-antioxidant status / copper / zinc)
A. A. ALTURFAN1, E. ZENGIN2, N. DARIYERLI3, E. E. ALTURFAN4, M. K. GUMUSTAS2,
E. AYTAC5, M. ASLAN6, N. BALKIS7, A. AKSU6, G. YIGIT3, E. USLU2, E. KOKOGLU2
1Vocational School Health Services, 2Department of Biochemistry, 3Department of Physiology,
4Department of Biochemistry, Faculty of Dentistry, Marmara University, Istanbul, Turkey
5Department of General Surgery, 6Department of Clinical Microbiology and Microbiology,
Istanbul University, Cerrahpasa Medical Faculty, Istanbul, Turkey
7Chemical Oceanography, Institute of Marine Sciences and Management, Istanbul University, Istanbul, Turkey
Recieved May 5, 2007. Accepted August 12, 2007.
Corresponding author: A. Ata Alturfan, Istanbul University, Vo-
cational School Health Services, Cerrahpasa Medical Faculty,
Kocamustafapasa, Istanbul, Turkey. Phone: (+90) 532 576 66 06;
fax: (+90) 212 5594792; e mail: firstname.lastname@example.org
Abbreviations: CAT – catalase(s), Cu – copper, GSH – glutath-
ione, MMI – methimazole, RNS – reactive nitrogen species, ROS
– reactive oxygen species, SOD – superoxide dismutase, T3 – tri-
iodothyroxin, T4 – thyroxin, TSH – thyroid-stimulating hormone,
Zn – zinc.
Folia Biologica (Praha) 53, 183-188 (2007)
Abstract. Thyroid hormones are associated with the
oxidative and antioxidative status of the organism.
Depression of metabolism by hypothyroidism has
been reported to decrease oxidant production and
thus protect tissues against oxidant damage. The
purpose of the present study was to investigate Zn and
Cu levels in MMI-induced hypothyroidism and to
show whether there is a connection between these
trace elements and the oxidant-antioxidant status in
experimental hypothyroidism. 3-Nitrotyrosine was
measured as a marker of nitro-oxidative stress. In or-
der to examine the antioxidant status of MMI-induced
hypothyroidism in rats, GSH and SOD levels were de-
termined as well. Significantly decreased 3-nitrotyro-
sine, Cu and Zn levels were observed in our experi-
mental model when compared with the controls. On
the other hand, GSH and SOD levels remained con-
stant. It may be suggested that Cu and Zn serve as
antioxidant molecules and exert their effects in an in-
direct manner to reduce oxidative stress in experi-
Hypothyroidism is a clinical entity resulting from defi-
ciency of thyroid hormones or, more rarely, from their im-
paired activity at the tissue level. In hypothyroidism, the
basal metabolic rate is decreased, as are other processes
dependent upon thyroid hormones (Hallengren, 1998;
Laurberg et al., 2005).
Oxidative stress, characterized by an elevation in the
steady-state concentration of reactive oxygen species
(ROS), has been implicated in a wide range of biologi-
cal and pathological conditions (Suntres and Lui, 2006).
Thyroid hormones are associated with the oxidative and
antioxidative status of the organism. Depression of me-
tabolism by hypothyroidism has been reported to de-
crease oxidant production and thus protect tissues
against oxidant damage. However, data on the oxidative
status of hypothyroidism are limited and controversial
(Isman et al., 2003; Sarandol et al., 2006).
Trace metals have been shown to influence hormones at
several levels, including hormone secretion and activity,
and binding to the target tissue (Aihara et al., 1984). Trace
elements such as zinc (Zn) and copper (Cu) possess a very
significant role in the regulation of many physiological
processes (Hughes and Saman, 2006). Cu enters cells by
membrane transporters and soluble transport proteins
called metallochaperones, and members of the cation
diffusion facilitator (CDF) family of transport proteins
transport Zn into and outside the cell (Gaither and Eide
2001; Rosenzweig, 2001). Human thyroid tissue con-
tains approximately 54.9 mg/kg Cu and 61 ppm Zn in it
(Boulyga et al., 1999; Yaman and Akdeniz 2004). Zn is
referred to as cofactor of many enzymes and has been
shown to be important in maintaining membrane func-
tion and integrity; it plays a major role in cellular signal-
ling. Moreover, zinc has been shown to have antioxidant
properties by protecting sulphydryl groups against oxi-
dation and inhibiting the production of ROS by transi-
tion metals (Bray and Bettger, 1990; Oteiza and Mac-
kenzie, 2005; Rubio et al., 2007). On the other hand, Cu
184 Vol. 53
undergoes redox-cycling reactions, constituents of oxidase
enzyme iron absorption. Furthermore, dietary Cu supple-
mentation may have beneficial effects by improving en-
dogenous antioxidant defences as well. Deficiency of Zn
and Cu has been observed to affect the endocrine system
adversely (Bordin et al., 1993; Galhardi et al., 2005;
Valko et al., 2005; Jones et al., 2006; Leach et al., 2006).
There are no conclusive references regarding Cu and
Zn influence on experimental hypothyroidism. In our
current study we hypothesized that Zn and Cu may sup-
port the antioxidant system in experimental hypothy-
Peroxynitrite (ONOO¯) is a reactive nitrogen species
(RNS) that has been found to cause lipid peroxidation and
cytotoxicity. ONOO¯ is a powerful oxidant which is high-
ly reactive toward biological molecules including protein
and non-protein sulphydryls, DNA, and membrane phos-
pholipids (Genovese et al., 2007). ONOO¯ is formed by
the rapid reaction of nitric oxide radical (NO˙) with super-
oxide radical (O2˙¯) (Whiteman et al., 2003; Radi, 2004).
Nonetheless, addition of peroxynitrite to biological fluids
leads to nitration of aminoacid residues, and the presence
of these has been widely used as a marker of peroxynitrite-
mediated (NO-dependent) damage in vivo (Kaur and Hal-
liwell, 1994). 3-Nitrotyrosine is generated when ONOO¯
is added to tyrosine itself, or to proteins containing ty rosine
residues (Daiber et al., 2004). Detection of protein 3-nitro-
tyrosine is regarded as a marker of nitro-oxidative stress
and is observed especially in inflammatory processes (Na-
viliat et al., 2005).
In aerobic cells, active oxygen species, e.g. O2˙¯ and
hydrogen peroxide (H2O2) are generated as by-products
of oxidative metabolism in mitochondria. These species
are toxic to biomembranes and eventually lead to per-
oxidation of lipids unless they are removed by free radi-
cal-scavenging enzymes. Antioxidant enzymes act to
scavenge free radicals by converting them to less harm-
ful molecules (Yilmaz et al., 2003). These might be re-
ferred to as enzymatic antioxidants such as superoxide
dismutase (SOD) and catalase (CAT) or metabolic anti-
oxidants like glutathione (GSH) (Hermans et al., 2007).
CAT represent the most important endogenous antioxi-
dant defence against ROS-induced damage of the cell
membrane. SOD protects tissues from oxygen free radi-
cals by catalyzing the removal of O2¯˙. Besides, CAT
were shown to be responsible for the detoxification of
significant amounts of H2O2 (Vergani et al., 2004; Nar-
vaez-Mastache et al., 2007). In blood plasma, these
chain-breaking antioxidants can trap free radicals di-
rectly, thereby interrupting chain-propagating reactions
(Koracevic et al., 2001). Furthermore, selenoproteins
are proteins that contain the selenocysteine form of sele-
nium. Although selenium has a variety of functions, its
antioxidant role has been the primary focus of research.
Glutathione peroxidase (GPX) and thioredoxin reduct-
ase (TR) play a major role in controlling two major re-
dox systems in the cell, i.e., the glutathione and thiore-
doxin systems. Selenium, therefore, as part of glutath-
ione peroxidase, is considered one of the antioxidant
nutrients and has interdependent roles with vitamin E,
iron (as catalase), and Zn and Cu (as superoxide dis-
mutase). Moreover, TR reduces thioredoxin, which in
turn is capable of controlling various cellular redox-re-
lated processes such as transcription (e.g., activation of
transcription factors), protein-DNA interactions, growth
control, and DNA synthesis. These antioxidants help
prevent the generation of free radicals, capable of re-
moving toxic hydroperoxides and decrease the risk of
oxidative damage to tissues. Additionally, other seleno-
proteins such as selenoprotein P and selenoprotein W
appear to play a role in oxidant defence as well (Glady-
shev et al., 1998; Holben and Smith, 1999).
The influence of thyroid failure on protein oxidation,
antioxidant status and trace elements have been studied;
however, not yet well fully understood. These findings led
us to explore Zn and Cu levels togerther with oxidant-
antioxidant system parameters in MMI-induced hy-
pothyroidism and to elucidate whether there is a connec-
tion between these trace elements and the oxidant-anti-
oxidant status in experimental hypothyroidism.
Material and Methods
In this study, 22 female Wistar Albino rats, 3 months
of age, weighing 151–174 g were used. Animals were
obtained from Istanbul University Animal Laboratory.
All experimental protocols were approved by the Ani-
mal Care and Use Committee.
The control group was fed ad libitum, while the ex-
perimental group received tap water plus MMI-added
fodder (75 mg/100 g) for 30 days, in order to induce hy-
pothyroidism (Dariyerli et al., 2004). MMI was given ac-
cording to the weight of the rats. Under urethane anaes-
thesia, blood samples were drawn from the heart into
plastic syringes between 08:00 and 10:00 am. Samples
were centrifuged at 2000 g for 10 min to obtain plasma.
Triiodothyroxin (T3), thyroxin (T4) and thyroid-stim-
ulating hormone (TSH) levels were measured by the
RIA method (Diagnostic Products Corporation, Los An-
geles, CA). The coat-A-count procedure is a solid-phase
radioimmunoassay where 125I-labelled T3, T4 and TSH
compete for a fixed time with T3, T4 and TSH in the sam-
ple for antibody sites. Radioactivity counting was per-
formed in a gamma counter (model 1185, Searle, Nu-
clear Chicago Division, Chicago, IL).
CuZn SOD activity measurement
CuZn SOD activity was determined by the method of
Sun et al. (1998) based on the inhibition of nitroblue
tetrazolium (NBT) reduction. The absorbance of the re-
duction product was read at 560 nm in a spectrophoto-
meter. One unit of SOD is defined as the amount of pro-
tein that inhibits the rate of NBT reduction by 50 %.
A. A. Alturfan et al.
Vol. 53 185
GSH levels were determined by the method of Beut-
ler et al. (1963).
The 3-nitrotyrosine quantification in plasma was
measured using an ELISA kit (ZID 7500 A, TCS Bio-
logical nitrotyrosine ELISA kit, Buckingham, UK). A
standard curve was constructed using serial dilutions of
nitrated BSA, which competed with immobilized nitrat-
ed proteins for the polyclonal anti-nitrotyrosine anti-
body. This method uses a rabbit anti-nitrotyrosine pri-
mary antibody and a donkey anti-rabbit IgG antibody
coupled to horseradish peroxidase as secondary anti-
body. The assay was performed as per manufacturers’
instructions (Bekpinar et al., 2005).
Cu and Zn measurement
One ml of serum was diluted to 10 ml with deionized
water. The test tube was placed in the auto-sampler car-
ousel after mild shaking. Serum samples were analysed
for Cu and Zn contents using an atomic absorption spec-
trophotometer. The measurement was carried out auto-
matically and the results are the average of two repli-
cates according to the standard addition calibration
method (Balkıs and Cagatay, 2001).
Differences between experimental groups were as-
sessed by the Mann-Whitney U test. The statistical soft-
ware package SPSS 10.0 (SPSS, Inc., Chicago, IL) was
used and P < 0.05 was considered significant.
The thyroid state of the animals is shown in Table 1.
A statistically significant increase in TSH and signifi-
cant decrease in T3 and T4 (P < 0.001) levels of the ex-
perimental group are evidence of induced hypothy-
At the end of the experimental period, the groups
were checked for the differences in weight and no sig-
nificant difference in weights was found (Table 1).
Significantly decreased plasma 3-nitrotyrosine, Cu
and Zn levels were observed in rats with hypothyroidism
when compared with the control group [(P < 0.01), (P <
0.01) and (P < 0.01), respectively]. Furthermore, no sta-
tistical significance was observed in the levels of SOD
and GSH levels [(P > 0.05), (P > 0.05)] when compared
with the control group.
One of the major effects of thyroid hormone is the
increment of mitochondrial respiration which results in
increased generation of ROS, leading to oxidative dam-
age to membrane lipids. There is a good deal of evi-
dence to indicate that metabolic depression brought
about by hypothyroidism is associated with a decrease
in free radical production and subsequent protection
against lipid peroxidation. This supports the notion that
the reduced demand for oxygen in hypothyroidism
might serve as a protective factor in tissue injury due to
reactive oxygen metabolites (Isman et al., 2003; Sener
et al., 2006a,b; Venditti and Meo, 2006). However, the
mechanism involved in this protective effect has not
been completely elucidated. Furthermore, MMI is sug-
gested to have antioxidant properties and act as a radical
scavenger, maintaining the GSH pool in the kidney of
rats (Sausen et al., 1992; Braunlich et al., 1997).
In our current work, the level of 3-nitrotyrosine which
reflects peroxynitrite-mediated oxidative damage in ex-
perimental hypothyroidism was apparently diminished.
Nitrosative stress occurs when the generation of RNS in
a system exceeds the system’s ability to neutralize and
eliminate them. Nitrosative stress may lead to nitrosyl-
Table 2. Plasma 3-nitrotyrosine, SOD, GSH, Cu and Zn levels of hypothyroid rats and the control group
3-nitrotyrosine (μM/L) SOD (U/ml) GSH (%mg) Cu (μg/dl) Zn (μg/dl)
Control (N = 11)
Hypothyroid (N = 11)
0.21 ± 0.02
0.18 ± 0.02*
19.54 ± 2.48
21.06 ± 2.62
27.01 ± 2.47
24.63 ± 1.90
131.56 ± 8.41
103.0 ± 10.27* 53.89 ± 4.94*
66.22 ± 8.73
Values are means ± S.E.M. (N = 11 in all groups) * P ≤ 0.05 was considered significant
Table 1. The results of the experimental procedure on rats
Controls (N = 11) Rats with hypothyroidism (N = 11) P value
T3 (ng/100 ml)
T4 (ng/100 ml)
161 ± 10
53.58 ± 5.69
4.31 ± 0.72
163 ± 11
23.58 ± 2.79
1.15 ± 0.28
1.28 ± 0.18
P > 0.05
P < 0.001
P < 0.001
P < 0.001
Values are means ± S.E.M. (N = 11 in all groups) * P ≤ 0.05 was considered significant
Antioxidants and Oxidants in Experimental Hypothyroidism
186 Vol. 53
ation reactions that can alter protein structure, thus in-
hibiting normal function. Tyrosine nitration is induced
by reactive nitrogen oxide species including ONOO¯,
which is synthesized by the reaction between superox-
ide anion O2˙ and NO˙. In hypothyroid rats, the de-
creased nitrosative stress is explained by the diminution
in oxygen consumption and O2˙ production (Tenorio-
Velazquez et al., 2005; Rubio et al., 2007). This supports
the idea that the reduced demand for oxygen in hypothy-
roidism might serve as a protective factor in tissue in-
jury due to reactive oxygen metabolites. As far as we are
concerned, one of the reasons for the diminution of pro-
tein oxidation in MMI-induced experimental hypothy-
roidism might root from the low level of secreted T3 and
T4 from the thyroid gland to the bloodstream. Another
protective mechanism might be due to the antioxidative
activity of MMI since sulphur-containing compounds
have been mentioned in the literature as antioxidants in
recent years (Nishimura et al., 2006). MMI was used as
an agent to induce hypothyroidsm; therefore, a possible
antioxidant effect of MMI should be taken into consid-
In a normal cell, there is an appropriate prooxidant:
antioxidant balance. However, this balance can be shifted
towards the prooxidants when the production of oxygen
species is increased greatly or when the levels of antioxi-
dants are diminished. In the literature, this state is called
“oxidative stress” and can result in serious cell damage if
the stress is massive or prolonged (Leach et al., 2006).
The mechanism of antioxidant system in MMI-in-
duced hypothyroid rats might be different. In our current
work, we hypothesized that Cu and Zn might be some-
how related with the oxidant-antioxidant status of the
organism in experimental hypothyroidism and this might
be reflected in the levels of these trace elements. We
found significantly decreased Cu and Zn levels in hy-
pothyroidism-induced rats compared with the controls.
Trace elements are known to influence many physio-
logical processes; however, evaluation of Zn and Cu as
antioxidants in experimental hypothyroidism is a novelty
of this study. Zn and Cu have not been shown to interact
directly with an oxidant species but rather prefer to exert
their effects in an indirect manner. In the present study,
GSH and SOD levels remained unchanged and seem not
to play a protective role in this experimental model.
Consequently, we may speculate that, in case of oxida-
tive stress, the first defence line could be Cu and Zn ele-
ments in bloodstream. There are a few suggestions on
this matter. Gibbs et al. (1985) and Bray et al. (1990)
have suggested two mechanisms for antioxidant proper-
ties of zinc. The first mechanism is about the protection
of sulphydryl groups against oxidation. The second
mechanism by which Zn may function as an antioxidant
molecule involves prevention of hydroxyl radical (OH˙)
and O2˙¯ production by transition metals. Very few sug-
gestions were made about the antioxidant properties of
Cu. Galhardi et al. (2005) investigated the effects of di-
etary Cu supplementation on the lipid profile and anti-
oxidant defences in serum of rats and reported that the
markers of oxidative stress, lipid hydroperoxide and li-
poperoxide, were decreased with Cu supplementation.
However, we should consider the role of low-molecular
forms of Cu in oxidative stress as well. OH˙ radicals
have attracted more interest than superoxide and H2O2
in biological systems, because they cause oxidative
damage to biomolecules. Although OH˙ generation re-
quires redox active transition metals such as Cu and Fe,
almost all cellular transition metal ions are tightly bound
to proteins. Cu-mediated oxidation of bio-molecules
such as DNA has been documented in detail. The pos-
sibility has been raised that the accumulated Cu metal-
lothioneins in Long-Evans rat (LEC) livers may partici-
pate in the copper Haber-Weiss reaction (Nakamura et
al., 1997). Zhang et al (2004) suggested that the home-
ostasis of metal ions in both serum and erythrocytes
could be more or less influenced by the altered thyroid
hormones, and they reported that serum Cu and Zn ex-
hibited significantly positive correlation with T3 and T4.
We also found decreased T3, T4 and Cu, Zn levels in
MMI-induced hyperthroid rats. These results may illus-
trate the protective effect of these trace elements as co-
factors of antioxidant enzymes in limiting oxidative
stress. The utilization of Cu and Zn by the CuZn SOD
enzyme may lower the levels of these trace elements in
plasma. In this regard, any significant modification of
the trace element status would lead to changes in the acti-
vity of antioxidants and have important consequences
on the susceptibility of tissues to oxidative stress.
In conclusion, our work suggests that there is equilib-
rium between oxidant and antioxidant systems in ex-
perimental hypothyroidism, where Cu and Zn have im-
portant roles in maintaining this equilibrium and sup-
porting the antioxidant functions. It is our belief that this
study will contribute positively to the studies dealing
with antioxidants, oxidative stress and experimental hy-
pothyroidism. Therefore, our current work will bring
different insight into the formation of new approaches in
prevention of pathologies resulting from oxidative dam-
age in hypothyroidism.
We thank Dr. Aybars Erozden for his assistance with
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