1. The effect of 3,5-diodo-L-thyronine and 3,5,3’-triiodo-L-thyronine on mitochondrial DNA integrity
Federica Cioffi 1, Pasquale Lasala 1, Angela Ziello 2, Liliana Iannucci 2, Antonia Giacco 1, Fernando Goglia 1and Antonia Lanni 2
1 University of Sannio, Benevento ITALY; 2 Second University of Naples, Caserta ITALY; federica.cioffi@unisannio.it; goglia@unisannio.it; antonia.lanni@unina2.it
Figure 1. Effect of T2 and T3 on mtDNA oxidative damage. (A) and (B) Mitochondrial DNA
damage was evaluated in the liver by amplifying long (13.4 Kb) and short (235 bp) regions
by QPCR; (C) Frequency of mtDNA lesion per 10 Kb per strand. Values are presented as
mean±SEM from 3 rats in each group. N control rats; N+T2 rats; N+T3 rats. *P<0.05 vs. N.
Figure 3. Effect of T2 and T3 on serum
levels of 8-OHdG. Values represent
means±SEM from 5 rats in each group.
N control rats; N+T2 rats; N+T3 rats.
*P<0.05 vs. N.
Fig. 2
Figure 2. Effect of T2 and T3 on mtDNA
content. Mitochondria DNA copy
number was assessed by real time PCR
in 10 ng liver gDNA using primer for
COII. The expression was normalized by
nuclear gene β-actin. Values are
presented as mean±SEM from 3 rats in
each group N control rats; N+T2 rats;
N+T3 rats. *P<0.05 vs. N.
Introduction
Thyroid hormones (THs) are a major factor controlling metabolic rate in virtually all cell
types in mammals. Due to their capacity to stimulate respiratory rate in mitochondria,
THs play a significant role in ROS production. But, as they simultaneously affect various
aspects of the oxidative stress, inducing different and even opposite effects, several
inconsistencies are found in the literature. Moreover, other iodothyronine, such as 3,5-
diiodo-l-thyronine (T2), has been shown to exert marked effects on energy metabolism
and on oxidative stress. Being the mitochondria the major site of ROS production and
target of TH, the purpose of this study was evaluate the effect of T2 and
triiodothyronine (T3) on rat liver mitochondrial DNA (mtDNA) oxidative damage and
repair.
Materials and Methods
Control rats, rats receiving a daily injection of 25 ug T2/100g BW (N+T2) for 1
week and rats receiving a daily injection of 15 ug T3/100 g BW (N+T3) for 1 week,
were used troughout. In liver, mtDNA oxidative damage [by measuring mtDNA
lesion frequency and expression of DNA polymerase γ (POLG), a selected
mtDNA repair enzyme], mtDNA copy number, mitochondrial biogenesis [by
measuring amplification of mtDNA/nDNA and expression of peroxisome
proliferator-activated receptor gamma coactivator 1 alpha (PGC1α)] and
oxidative stress [by measuring serum levels 8-hydroxy-2’-deoxyguanosine (8-
OHdG)] were detected.
Results/Discussion
T2 reduces mtDNA lesion frequency, increases expression of POLG, does not change
mtDNA copy number and expression of PGC1α, and does not alter serum levels of 8-
OHdG. Therefore, T2 by stimulating the major mtDNA repair enzyme maintains
genomic integrity. Like T2, T3 reduces mtDNA lesion frequency, but increases serum
levels of 8-OHdG and decreases expression of POLG. Moreover, as expected, it increases
mtDNA copy number and expression of PGC1α. Thus, in T3 treated rats the increase of
8-OHdG and the decrease of POLG indicate that there is greater oxidative damage
and that the reduced mtDNA lesion frequency might be a consequence of increased
mitochondrial biogenesis.
Figure 5. Effect of T2 and T3 on POLG expression. (A) POLG mRNA expression was
measured by real time PCR using β-actin as an internal control; (B) Protein levels of
POLG were quantified by Western Blot using β-actin as the loading control. Each
value represents the mean±SEM from 3 rats in each group. N control rats; N+T2 rats;
N+T3 rats. *P<0.05 vs. N.
Figure 4. Effect of T2 and T3 on PGC1a expression. (A) PGC1α mRNA expression was
measured by real time PCR using β-actin as an internal control; (B) Protein levels of
PGC1a were quantified by Western Blot using β-actin as the loading control. Each value
represents the mean±SEM from 3 rats in each group. N control rats; N+T2 rats; N+T3 rats.
*P<0.05 vs. N.
Conclusions
Taken together these data
demonstrate that both T2 and T3
are able to reduce mtDNA
oxidative damage but they act via
different mechanisms
Fig. 3
Fig. 5
A) B)
A) B)
Fig. 4
Fig. 1
A) B)
C)
1) Villanueva I, Alva-Sánchez C, Pacheco-Rosado J., The role of thyroid hormones as inductors of oxidative stress and neurodegeneration. Oxid Med Cell Longev. 2013; 2) Janine H. Santos, Joel N. Meyer, Bhaskar S. Mandavilli, Bennett Van Houten. Quantitative PCR-Based Measurement of Nuclear and Mitochondrial DNA Damage and Repair in Mammalian Cells. DNA Repair Protocols, Methods in Molecular Biology, Volume 314, 2006, pp 183-199; 3) Mollica MP, Lionetti L,
Moreno M, Lombardi A, De Lange P, Antonelli A, Lanni A, Cavaliere G, Barletta A, Goglia F. 3,5-diiodo-l-thyronine, by modulating mitochondrial functions, reverses hepatic fat accumulation in rats fed a high-fat diet. J Hepatol. 2009 Aug;51(2):363-70.
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