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First published online March 12, 2009
Journal of Experimental Biology 212, 986-993 (2009)
Published by The Company of Biologists 2009
doi: 10.1242/jeb.021808
T3 and the thyroid hormone β-receptor agonist GC-1 differentially affect metabolic capacity and oxidative damage in rat tissues
1 Dipartimento delle Scienze Biologiche, Sezione di Fisiologia,
Università di Napoli, 80134 Napoli, Italy
2 Dipartimento di Scienze dell'Uomo e dell'Ambiente (G.C.), University of Pisa,
Pisa 56126, Italy
3 Department of Toxicology, Oncology, and Molecular Pathology Unit, University
of Cagliari, 09124 Cagliari, Italy
4 Department of Physiology and Pharmacology and Department of Cell and
Developmental Biology, Oregon Health and Science University, Portland, OR
97239, USA
* Author for correspondence (e-mail: venditti{at}unina.it)
Accepted 6 January 2009
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Summary |
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 TOP Summary INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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Key words: thyroid hormone, thyroid hormone agonist, oxidative metabolism, oxidative damage
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INTRODUCTION |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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), and
increases cardiac contractile performance
(Buccino et al., 1967) and
heart rate (Di Meo et al.,
1994) in response to the demand for greater tissue perfusion.
Thyroid hormone is used in the treatment of hypothyroidism and some thyroid
cancers. Because of its metabolic effects, therapeutic thyroid hormone
administration might also be extended to other disorders such as obesity
(Krotkiewsky, 2002) and dyslipidaemia
(Hansson et al., 1983).
However, the therapeutic utilization of T3 might be accompanied by
harmful side effects, particularly cardiac dysfunction
(Klein and Ojamaa, 2001).
Thus, there is interest in developing analogues of T3 that elicit
desirable but not unwanted effects.
It is theoretically possible to discriminate among the T3
effects since they are due to the interaction of the hormone with two subtypes
of nuclear receptor, TR
and TRβ, which selectively mediate
tissue-specific thyroid hormone responses
(Forrest and Vennström,
2000). TR mediates T3 effects on heart rate and
modulates body temperature, whereas TRβ mediates the cholesterol-lowering
and thyroid-stimulating hormone (TSH)-suppressant effects of T3
(Forrest and Vennström,
2000). Therefore, the development of T3 analogues that
bind preferentially to TRβ has been considered a possible strategy to
obtain beneficial T3 effects whilst avoiding the harmful ones.
Recently, the availability of
3,5-dimethyl-4-(4'-hydroxy-3'-isopropylbenzyl)phenoxy acetic acid
(GC-1), a TRβ-selective agonist, has allowed discrimination amongst the
different effects of thyroid hormone. Indeed, GC-1 lowers serum cholesterol
and triglycerides in measure equal to or greater than T3, without
significant stimulation of heart rate
(Trost et al., 2000). The
observed preferred accumulation of GC-1 in the liver vs the heart
probably also contributed to the marked lipid-lowering effect in the absence
of an effect on heart rate (Trost et al.,
2000). More recently, in euthyroid monkeys a significant
GC-1-induced cholesterol lowering and body mass loss has been observed,
resulting from a modest increase in metabolic rate
(Grover et al., 2004). A
moderate increase in O2 consumption and a large decrease in plasma
cholesterol was also found in cholesterol-fed euthyroid rats
(Grover et al., 2004). The
above results suggest the possibility that selective agonists, such as GC-1,
can be used for the treatment of obesity and hypercholesterolaemia even though
further studies are necessary to establish the cellular mechanisms of the
metabolic rate regulation. It is well known that the T3-induced
increase in basal metabolic rate is dependent on accelerated O2
consumption in target tissues, secondary to an increased content of
mitochondrial respiratory chain components
(Nishiki et al., 1978;
Horrum et al., 1985). This
metabolic response is accompanied, as a side effect, by an enhanced generation
of reactive oxygen species (ROS) leading to oxidative stress development in
rodent tissues as well as in hyperthyroid patients, as documented by enhanced
levels of indicators of lipid and protein oxidation (reviewed by
Venditti and Di Meo, 2006;
Fernández et al.,
2006). Because the ability of a substance to induce conditions of
oxidative stress represents a contraindication for its therapeutic
utilization, the necessity to investigate the relationship between TRβ
activation and both aerobic metabolism and oxidative damage of the tissues is
apparent. To shed light on this subject, we compared the effects of
T3 and GC-1 treatment of hypothyroid rats on the aerobic metabolism
and oxidative damage of liver, heart and skeletal muscle. Accordingly, we
determined the cytochrome oxidase (COX) activity, and oxygen consumption in
the presence of complex I- and complex II-linked substrates (pyruvate/malate
and succinate, respectively). We also determined hydroperoxide and
protein-bound carbonyl levels as markers of oxidative damage to lipids and
proteins, respectively. Finally, to evaluate the possible impact of the
antioxidant defence system on the extent of tissue oxidative damage in
T3- and GC-1-treated rats, we determined: (i) activities of
antioxidant enzymes, such as glutathione peroxidase (GPX) and glutathione
reductase (GR); (ii) levels of free radical scavengers, such as vitamin E,
coenzyme Q (CoQ) and reduced glutathione (GSH); and (iii) the in vitro
tissue susceptibility to oxidants.
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MATERIALS AND METHODS |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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). The
response to oxidative stress was determined using the reagents and
instrumentation of the commercially available Amerlite System (Ortho-Clinical
Diagnostics, Milano, Italy). Serum levels of free triiodothyronine
(FT3) and thyroxine (FT4) were determined using a
commercially available RIA kit (DiaSorin, Salluggia, Italy). Serum cholesterol
levels were measured using a commercial kit available from Pokler Italia
(Genova, Italy).
Animals
The experiments were carried out on 70-day-old male Wistar rats (Rattus
norvegicus albius Berkenkault 1769), supplied by Nossan (Correzzana,
Italy). From day 49, animals were randomly assigned to one of four groups:
control euthyroid rats (C); hypothyroid rats (H); and hypothyroid rats treated
with T3 (H+T3) or GC-1 (H+GC-1). In H rats, both thyroid
and deiodinase activities were chronically inhibited by intraperitoneal
administration of propylthiouracil (PTU; 1 mg 100 g–1 body
mass, once per day for 3 weeks), together with administration of iopanoic acid
(IOP) given 10, 13, 16 and 19 days after the first PTU injection. Some rats
that were subject to the same treatment also received daily intraperitoneal
injections of 15.36 nmol of T3 per 100 g body mass for 10 days. The
above experimental protocols induce hypothyroidism and hyperthyroidism,
respectively, in the laboratory rat
(Moreno et al., 1997).
Moreover, the T3 dose used to elicit the hyperthyroid state
increases resting metabolic rate (Venditti
et al., 2004) and induces tissue oxidative stress
(Venditti et al., 1997) in
rat. To compare the T3 and GC-1 effects on metabolic and oxidative
processes, the other rats were treated with 10 daily equimolar doses of GC-1
(15.36 nmol 100 g–1 body mass). We chose hypothyroid rats for
the treatment with the agonists to obtain animals whose serum contains only
T3 or GC-1. All rats were kept under the same environmental
conditions and were provided with water ad libitum and a commercial
rat chow diet (Nossan).
The treatment of animals in these experiments was in accordance with the guidelines set forth by the University's Animal Care Review Committee.
Tissue preparation
After a 12 h overnight fast, the resting metabolic rate of the animals was
measured using an open circuit indirect calorimetric system (Panlab Gas
Analyzer LE 405, Madrid, Spain). The animals were then anaesthetized with
Ethrane (Abbot, Aprilia, Italy) and subjected to electrocardiographic
recording. Arterial blood samples were collected and later analysed to
determine serum levels of FT3, FT4 and cholesterol.
While still under anaesthesia, the animals were killed by decapitation and
tissues (liver, heart and gastrocnemious muscle) were rapidly excised and
placed into ice-cold homogenization medium (HM: 220 mmol l–1
mannitol, 70 mmol l–1 sucrose, 1 mmol l–1
EDTA, 0.1% fatty acid-free albumin, 10 mmol l–1 Tris, pH
7.4). The heart great vessels and valves were trimmed away and the ventricles
and atria were cut open and rinsed free of blood. Muscle and liver were freed
from connective tissue. Then the tissues were weighed, finely minced, and
washed with HM. Muscular tissue fragments were incubated for 5 min with HM
containing 0.1 mg ml–1 nagarse and washed. Finally, all
tissues were gently homogenized (20% w:v) in HM using a glass
Potter–Elvehjem homogenizer set at a standard velocity (500 r.p.m.) for
1 min. Tissue homogenates were used for analytical procedures.
Analytical procedures
Cytochrome c oxidase activity was determined polarographically at
30°C using a Gilson glass respirometer equipped with a Clark oxygen
electrode (Yellow Springs Instruments, OH, USA) following the procedure of
Barré and colleagues (Barré
et al., 1987). COX is the final respiratory enzyme, and its in
vitro activity has been positively correlated with maximal oxygen
consumption (Simon and Robin, 1981) so that such activity can be used as a
measure of the aerobic metabolic capacity of tissues.
Oxygen consumption was monitored at 30°C with the above Gilson respirometer in 1.6 ml of incubation medium (145 mmol l–1 KCl, 30 mmol l–1 Hepes, 5 mmol l–1 KH2PO4, 3 mmol l–1 MgCl2, 0.1 mmol l–1 EGTA, pH 7.4) with 50 µl of homogenate and succinate (10 mmol l–1), plus 5 µmol l–1 rotenone, or pyruvate/malate (10/2.5 mmol l–1) as substrates, in the absence (state 4) and in the presence (state 3) of 500 µmol l–1 ADP. Furthermore, the ratio between state 3 and state 4 respiration rates (respiratory control ratio, RCR) was calculated. Basal (state 4) respiration represents a compensatory response to the leak of protons back into the mitochondrial matrix, so that changes in its rate give information on the basal proton conductance of the inner mitochondrial membrane. Conversely, rates of ADP-stimulated (state 3) respiration supply information on the rate of oxidative phosphorylation. The respiration measurements performed in the presence of pyruvate/malate and succinate allowed us to discriminate among effects concerning different segments of the mitochondrial electron transport chain.
The extent of the peroxidative processes was determined by measuring the
level of lipid hydroperoxides (HPs) (Heath and Tappel, 1976). Tissue protein
oxidation was assayed by the reaction of 2,4-dinitrophenylhydrazine with
protein carbonyls (Reznick and Packer,
1994).
GPX activity was assayed at 37°C
(Flohé and Günzler,
1984), with H2O2 as the substrate. GR
activity was measured at 30°C (Carlberg
and Mannervik, 1985).
Ubiquinols (CoQH2) from 0.5 ml of 10% homogenate were oxidized
to ubiquinones (CoQs) with 0.5 ml of 2% FeCl3 and 2.0 ml of
ethanol. The total content of CoQs (CoQH2 + CoQ) was then
determined by HPLC (Lang et al.,
1986). Vitamin E content was determined using the HPLC procedure
of Lang and colleagues (Lang et al.,
1986). GSH concentration was measured in homogenates as described
by Griffith (Griffith,
1980).
The response to oxidative challenge was determined as previously described
(Venditti et al., 1999). In
brief, samples of 10% (w/v) homogenates were obtained by diluting the 20%
homogenates with equal volumes of 0.2% Lubrol in 15 mmol l–1
Tris, pH 8.5. Several dilutions of the samples up to a tissue concentration of
0.002% were prepared in 15 mmol l–1 Tris (pH 8.5). The assays
were performed in microtitre plates. Enhanced chemiluminescence reactions were
initiated by addition of 250 µl of the reaction mixture to 25 µl of the
samples. The reaction mixture was obtained by mixing solutions containing
substrate in excess and signal-generating reagents in buffer at pH 8.6 (Vitros
Signal Reagent, Ortho Clinical Diagnostics, High Wycombe, Bucks, UK). The
plates were incubated at 37°C for 30 s under continuous shaking and then
transferred to a luminescence analyser (Amerlite Analyzer). The emission
values were fitted to dose–response curves using the statistical
facilities of the Fig. P graphic program (Biosoft, Cambridge, UK).
Data analysis
The data, expressed as means ± s.e.m., were analysed using a one-way
variance method (ANOVA). When a significant F ratio was found, the
Student–Newman–Keuls multiple range test was used to determine the
statistical significance between means. Probability values
(P)<0.05 were considered significant. In
Fig. 2 the results of the
experiments are presented as sample curves.
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RESULTS |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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Oxygen consumption
Data on rates of succinate-supported oxygen consumption by tissue
homogenates are reported in Table
2. In liver, such rates were lower in the hypothyroid than in the
euthyroid group during both state 4 and state 3. Conversely, O2
consumption rates were increased by T3 and GC-1 treatment during
both respiration states, with the highest rates reached in the H+T3
group. The highest values of the RCR were also found in the H+T3
preparations, while the lowest ones were found in the H and H+GC-1
preparations. In heart, both state 4 and state 3 respiration rates were
decreased by PTU+IOP treatment and increased by T3 treatment.
Following GC-1 treatment respiration rates were not significantly increased,
remaining lower than control values during state 4. The RCR values were not
significantly modified by the treatments. In muscle, respiration rates during
state 4 and state 3 were decreased by PTU+IOP treatment. Treatment of
hypothyroid rats with T3 and GC-1 increased both state 4 and state
3 respiration in different measure so that they were higher than in controls
only in H+T3 preparations. RCR values were significantly higher in
H+T3 than in other groups.
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Data on rates of pyruvate/malate-supported oxygen consumption by tissue homogenates are reported in Table 3. In liver, during both state 4 and state 3 rates were decreased by PTU+IOP treatment and increased by T3 and GC-1 treatment. Although the greater changes were elicited by T3, in both H+T3 and H+GC-1 preparations respiration rates were higher than in the C group. RCR values were not affected by the treatments. In heart, state 4 and state 3 respiration rates were decreased by PTU+IOP treatment and not modified by subsequent treatment with GC-1. Conversely, respiration rates were increased by T3 treatment, but only during state 3 were they higher in the H+T3 than in the C group. RCR values were not affected by the treatments. In muscle, respiration rates were decreased during both state 4 and state 3 by PTU+IOP treatment. T3 and GC-1 treatments caused different increases in such rates so that they were not different in H+GC-1 and C groups, and were higher in H+T3 than in all the other groups. RCR values were different in the C, H and H+GC-1 groups, and were highest in the H+T3 group.
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Oxidative damage
As shown in Table 4,
treatment with PTU+IOP reduced lipid peroxide and protein-bound carbonyl
levels in liver and muscle, and carbonyl levels in heart. After T3
treatment, tissue levels of both parameters significantly increased compared
with hypothyroid and euthyroid values even though lower increases were found
for lipid peroxide levels. GC-1 treatment was less effective than
T3 at increasing hydroperoxide and carbonyl levels, which resulted
in higher than hypothyroid values in all tissues, but higher than euthyroid
values only in liver.
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Antioxidants
The effects of treatment with PTU+IOP, T3 and GC-1 on tissue
antioxidants are reported in Table
5. In liver, GPX activity was not modified by PTU+IOP treatment,
and was significantly increased by T3 and GC-1 compared with the
euthyroid values. GR activity, which was initially lower in hypothyroid than
in euthyroid rats, was higher than both the euthyroid and hypothyroid values
after T3 treatment, whereas after GC-1 treatment it was not
different from either. Vitamin E content, which was decreased by PTU+IOP
treatment, was restored to the euthyroid level by GC-1 and greatly increased
by T3. CoQ10 levels, which were not different in euthyroid and
hypothyroid rats, were increased by the treatments. However, CoQ9 levels were
lower in GC-1-than in T3-treated rats. GSH content was increased in
hypothyroid rats and was decreased by agonist treatment so that it was lower
in H+T3 and H+GC-1 rats than in euthyroid controls and reached the
lowest value in T3-treated rats.
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In heart, following PTU and IOP treatment, GPX and GR activities were decreased compared with euthyroid values, which were restored by both T3 and GC-1 treatment. Vitamin E content was lower in the hypothyroid than in the euthyroid group. Treatment with T3 or GC-1 was associated with different increases in vitamin content so that in comparison with control values it was higher in H+T3 and lower in H+GC-1 rats. CoQ9 and CoQ10 levels were not different in euthyroid and hypothyroid rats and were increased by T3 and GC-1 treatments, reaching the highest value in H+T3 rats. GSH levels, which were decreased by treatment with PTU and IOP, were further decreased by T3 but not by GC-1 treatment.
In muscle, GPX activity was not modified by the different treatments, whereas GR activity was higher in H+T3 and H+GC-1 than in C and H groups. Vitamin E content was decreased by PTU+IOP treatment and was differently increased by agonist treatment. Thus, vitamin content was higher in H+T3 and H+GC-1 than in the other groups and reached the highest value in the T3-treated group. CoQ9 and CoQ10 levels were not significantly affected by PTU+IOP treatment and were increased by agonist treatment. CoQ9 reached the highest value in the H+T3 group, whereas CoQ10 levels were not significantly different in T3- and GC-1-treated rats. GSH levels were lower in the hypothyroid than in the euthyroid group. Treatment with T3 but not GC-1 caused a further decrease in such levels.
Response to oxidative challenge
The relationship between light emission (E) and homogenate
concentration (C) was described by the equation
E=aC/exp(bC), where a and b are
two constants (Venditti et al.,
1999). A careful examination of this function shows that
b is the inverse of the homogenate concentration at the emission
maximum (Emax), which, in turn, is determined by
a and b values
(Emax=a/eb). As shown by the
dose–response curves reported in Fig.
2, the emission levels in general and Emax in
particular were not affected by PTU+IOP, increased by T3, and
differently affected by GC-1 treatment. These qualitative evaluations are
confirmed by the results reported in Table
6, which show that GC-1 treatment increases
Emax levels to a smaller extent than T3
treatment in the liver and its effects are not significantly different from
T3 treatment in the muscle, whereas it does not affect
Emax levels in the heart.
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Examination of the parameters determining light emission shows that significant increases in the value of a are induced in all preparations by T3 but not by GC-1. Significant decreases in the b values in comparison to euthyroid values were found after agonist treatment only in liver and muscle preparations.
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DISCUSSION |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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; Venditti et al.,
2004), whereas no comparable data are available in the literature
concerning the GC-1 effect. On the other hand, significant differences were
also found in the responses to T3 and GC-1 of other specific
thyroid hormone-responsive parameters. Thus, heart mass was increased by
T3, but not by GC-1, whereas heart mass/body mass ratio, heart rate
and resting metabolic rate were increased by both agonists, but in smaller
measure by GC-1, so that their values in the H+GC-1 group were not higher than
those found in the euthyroid group.
In agreement with the observation that the increased capacity for heat production, induced by T3 administration, arises from an increased capacity for oxidative metabolism of target tissues, we found increased COX activity in all examined tissues resulting from T3 and GC-1 treatment of hypothyroid rats. However, whereas the relative increases induced by T3 in liver, heart and muscle were similar enough (62.4%, 48.3% and 68.4%, respectively) those induced by GC-1 were remarkably different (54.8%, 14.7% and 30.4%, respectively), so that only liver COX activity underwent similar changes after T3 and GC-1 treatment and was higher in GC-1-treated than in euthyroid control rats.
The potential mechanism underlying these and other responses to GC-1 is
likely to be linked to the differences in GC-1 affinity for the T3
nuclear receptor isoforms, as well as the different GC-1 and TRβ isoform
distribution in the various tissues. Studies in cell culture have shown that
T3 and GC-1 have a similar binding affinity for the TRβ
subtype, whereas T3 affinity for the TR subtype is
approximately 10 times greater than the GC-1 affinity
(Chiellini et al., 1998). It
has also been shown that, whereas TRβ accounts for 80% of the TR binding
in the liver (Schwartz et al.,
1992), close to half of the T3 receptors in the heart
are of the TR subtype (Schwartz et
al., 1992), which is also the predominant isoform in the skeletal
muscle (White et al., 2001).
Moreover, measurement of GC-1 and T3 in mouse plasma and tissues
has revealed significant differences in their organ distributions
(Trost et al., 2000). The
tissue to plasma ratios in the liver and muscle are of the same order of
magnitude for GC-1 and T3, whereas in the heart they are 30 times
higher for T3 than for GC-1. If these results are the same in rats,
they may explain why the GC-1-induced changes in aerobic capacity are lower in
heart than in muscle, although the distribution of the TRβ isoform is
higher in cardiac muscle. If so, the ability of GC-1 treatment to restore
euthyroid values of heart rate, heart mass/body mass ratios, and aerobic
capacity in hypothyroid rats might reflect indirect effects resulting from
increased oxygen consumption in other tissues.
Further information on the respiratory characteristics of the tissues was
obtained by measuring the in vitro oxygen consumption of their
homogenates. As expected, we found that in all tissues basal and
ADP-stimulated O2 consumption decreased in hypothyroid and
increased in T3-treated rats. Conversely, tissue-dependent
differences were shown in the respiratory responses of hypothyroid tissues to
GC-1 treatment. With the exception of a report dealing with UCP1 stimulation
in mouse brown adipose tissue (Ribeiro et
al., 2001), the effects of GC-1 on basal and inducible
mitochondrial proton conductance have not been studied. Therefore, we are not
able to explain the limited changes in the state 4 respiration induced by GC-1
in liver and muscle preparations. The failure of GC-1 to induce changes in
state 3 respiration in heart homogenate was consistent with its poor influence
on COX activity. Thus, the GC-1-induced increases in liver and muscle
respiration agreed well enough with the respective increases in COX activity.
However, in both tissues state 3 respiration underwent different increases in
the presence of complex I- and complex II-linked substrates. For liver, they
were about 88% and 67%, respectively, whereas for muscle they were 36% and
12%, respectively. The finding that T3 induces a similar
stimulation of state 3 respiration in the presence of pyruvate/malate and
succinate (177% and 180% for liver, and 87% and 83% for muscle) suggests that
the components of the respiratory chain do not respond as a unit to GC-1
stimulation unlike the case for T3 stimulation.
It is now widely accepted that in living systems normal metabolic
processes, that are essential to the cells, lead to the formation of ROS. The
main biological process leading to ROS generation is electron transport within
the inner mitochondrial membrane. Normally, oxygen is reduced in mitochondria
to form water by concerted four-electron transfer. However, oxygen can also
undergo univalent reduction by a one-electron transfer, which allows the
formation of superoxide anion radical
(
), hydrogen peroxide
(H2O2) and hydroxyl radical
(·OH), which can oxidatively damage biological
molecules. Aerobic organisms are provided with a system of biochemical
defences to neutralize the oxidative ROS effects, but when free radical
generation exceeds the antioxidant capacity of cells oxidative stress
develops. This phenomenon has been related to many pathological conditions,
but it can also occur as a result of hypermetabolic state induced by normal
physiological activities such as physical exercise
(Barja de Quiroga, 1992). A
considerable body of evidence attests that a hypermetabolic state typical of
hyperthyroidism also results in oxidative injury secondary to increased ROS
production and decreased antioxidant capacity in rodent tissues
(Venditti and Di Meo, 2006;
Fernández et al.,
2006). It is also well established that thyroid hormone induces
oxidative stress in humans. Clear evidence for this was provided by the
finding of increased circulating levels of H2O2 and
indices of peroxidative damage in hyperthyroid patients
(Venditti and Di Meo, 2006;
Fernández et al.,
2006). Moreover, it was suggested that ROS play a role in myopathy
and cardiomyopathy, which are the major complications of hyperthyroidism
(Asayama and Kato, 1990).
Because GC-1 is able to induce changes in basal metabolic rate and aerobic
metabolism of thyroid hormone target tissues, it raises the question of the
extent of oxidative damage associated with metabolic changes. Thus, we
investigated the relative effects of GC-1 and T3 on tissue
oxidative damage. Our results confirm a previous report indicating that
T3 treatment of hypothyroid rats induces oxidative stress in liver,
heart and skeletal muscle (Venditti and Di
Meo, 2006). Administration of equimolar doses of GC-1 increases
tissue markers of oxidative damage to a lesser extent than T3
treatment so that only the oxidative damage to liver lipids is significantly
higher in the H+GC-1 than in the C group. Oxidative damage to lipids and
proteins depends on different biochemical characteristics, such as the degree
of unsaturation of hydrocarbon chains and the content of amino acids able to
generate carbonyl groups, respectively. Thus, it is not surprising that in all
tissues the two agonists differentially damage lipids and proteins. It is less
clear how GC-1 induces oxidative damage in cardiac tissue, which is much less
prone to its action. One possibility is that the oxidative damage of the
cardiac muscle depends on its activity. This idea is indirectly supported by
the observation that the increase in cardiac activity, induced by acute
exercise, is associated with enhanced free radical generation along with
augmented lipid peroxidation (Kumar et
al., 1992).
To evaluate the impact of antioxidant protection changes on the different
extent of tissue oxidative damage in T3- and GC-1-treated rats, we
determined the activities of antioxidant enzymes and the levels of free
radical scavengers, which are major components of the antioxidant defence
system. Indeed, the combined action of GPX and GR is the main determinant of
tissue content of GSH, the most abundant cellular thiol. Vitamin E is the main
chain-breaking antioxidant preventing free radical-initiated peroxidation
(Tappel, 1972). CoQ carries
out an antioxidant function in cell membranes, which can be ascribed to a
synergism with vitamin E (Kagan et al.,
1990) or a direct radical scavenging activity
(Ernster et al., 1992). Our
results show that GSH levels are higher in H+GC-1 than in H+T3
preparations. Because the fall in GSH levels after a damaging treatment can be
considered an index of oxidative stress it is not surprising that the fall is
greater in H+T3 preparations, which suffer more extensive oxidative
damage. Conversely, GPX and GR activities and liposoluble antioxidant levels
do not seem to be related to the different gravity of oxidative stress found
in rats treated with T3 or GC-1 and do not supply information on
the protective efficacy of the antioxidant system of the tissues. On the other
hand, even determination of the concentrations of all ROS scavengers and the
activities of all antioxidant enzymes should supply only a partial evaluation
of such an efficacy, which greatly depends on the system's ability to
co-ordinate and integrate the function of the various antioxidants.
However, we obtained some information on the effectiveness of the
antioxidant system by a method able to evaluate tissue sensitivity to
oxidative processes (Venditti et al.,
1999). In this method the extent of oxidative changes occurring in
conditions leading to increased in vivo ROS production is obtained by
measuring the levels of light emission resulting from in vitro
exposure to H2O2 of tissue homogenates.
Because the level of light emission, and particularly the emission maximum,
can be considered an index of the susceptibility of the preparations to
oxidative challenge (Di Meo et al.,
1996), our results reveal an increased susceptibility following
both treatments, but in smaller measure with GC-1.
Analysis of the parameters affecting the emission levels shows that the
lower levels found in H+GC-1 rats are due mainly to lower values of the
parameter a, whereas a marginal contribution is supplied by changes
in the value of parameter b. It has been shown that the values of
parameters a and b are dependent on the concentration of
substances able to induce and inhibit, respectively, the
H2O2-promoted luminescence reaction
(Venditti et al., 1999). It
has also been shown that substances able to induce the luminescence reaction
include Fe2+ complexes, such as the haemoproteins, which promote
the conversion of H2O2 into reactive
·OH radicals via the Fenton reaction
(Halliwell and Gutteridge,
1990), whereas substances inhibiting the luminescence reaction
include antioxidants able to prevent the formation of or interact with
·OH radicals.
Thus, the aforementioned results supply indirect evidence that a smaller increase in tissue levels of respiratory mitochondrial chain components is responsible for both a lower oxidative capacity and a lower degree of oxidative damage induced by GC-1 in rat tissues. Conversely, the changes in antioxidant capacity are too little to distinguish the oxidative effects of T3 and GC-1 treatments. However, further studies are necessary to establish whether a lower generation of ROS contributes to make the extent of oxidative damage smaller in H+GC-1 than in H+T3 preparations.
In summary, the results reported in the present paper indicate that GC-1 induces some beneficial effects of T3, such as a lowering of the plasma cholesterol level, and causes a moderate increase in basal metabolic rate due to an enhancement of target tissue aerobic metabolism lower than that elicited by T3. It is important that GC-1 can elicit these effects at doses that are able to limit the increase in heart rate and other detrimental effects, such as an enhancement of tissue oxidative damage and susceptibility to oxidants, which accompany treatment with equimolar doses of T3. Thus, our paper shows that GC-1 has promising properties for a possible therapeutic utilization. However, further studies are necessary to establish whether GC-1 can have unforeseen toxic effects for mammalian species and in particular for humans, in which it has never been tested.
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Footnotes |
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References |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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