Measurement of the 4834 bp mitochondrial DNA deletion level by Real Time PCR in aging rat liver subjected or not to caloric restriction diet
P. Cassano, A.M.S. Lezza, C. Leeuwenburgh, P. Cantatore, M.N. Gadaleta
Department of Biochemistry and Molecular Biology, University of Bari, via Orabona 4 70126 Bari, Italy
Aging is a fascinating and highly important topic because of its great
social relevance and scientific complexity. One of the most important
theories to explain this multifactorial process is the free radical
theory. Such theory involves the damaging role of reactive oxygen
species (ROS) on mitochondrial molecular components as lipids, proteins
and mitochondrial DNA (mtDNA). Several studies have demonstrated an
age-related accumulation of the number of deleted species of mtDNA as
well as of the amount of a specific 4834 bp mtDNA deletion, homologue
of the human mtDNA "common deletion", in different tissues of rat
(liver, brain and skeletal muscle) (1-3). The tissue-specificity of
age-related mechanisms is supported by the different degrees of effect
of the so-far only known treatment able to delay the aging degeneration
namely caloric restriction (CR). CR treatment has been reported to
decrease the number of mtDNA deleted species in rat skeletal muscle
(3), but nothing is known about in other tissues. Therefore, we decided
to evaluate by quantitative Real Time PCR the level of the 4834 bp
mtDNA deletion in tissues of aging rats treated and not with CR. The
chosen tissues include some clearly sensitive to the effect of CR in
reducing mitochondrial oxidative stress that is liver and skeletal
muscle (4), while brain has also been included in our study because of
the CR efficacy shown by microarray approach (5). Data concerning the
level of the 4834 bp mtDNA deletion in liver from adults fed ad libitum
(AL), old fed ad libitum (OL) and old lifelong 40% calories-restricted
(OR) Fischer animals are presented. The level of the 4834 bp mtDNA
deletion has been determined with respect to the mitochondrial D-loop
level, using specific primers and TaqMan probes for each target. The
method has been validated by measurements in previously assayed samples
and evaluating the equal reaction efficiency of the two amplicons. We
found an age-related increase of the deletion level in OL animals (the
mean value is a two-fold increase) that was reversed and brought back
to the adult level by long term CR.
References:
1.Gadaleta M.N., Rainaldi G., Lezza A.M.S., Milella F., Fracasso F., Cantatore P. (1992), Mutat. Res., 275: 181-193.
2.Gadaleta M.N., Rainaldi G., Lezza A.M.S., Marangi L.C., Milella F., Daddabbo L., Fracasso F., Loguercio Polosa P., Cantatore P. (1995), In: Progress in Cell Research (F. Palmieri et al. eds), Elsevier Science, Amsterdam, vol. 5 pp. 231-235.
3.Aspnes L., Lee C.M., Weindruch R., Chung S., Roecher E., Aiken J. (1997), FASEB J., 11, 573-581.
4.Gredilla R., Barja G., Lopez-Torres M. (2001), J. Bioenerg. Biomembr., 33: 279-287
5.Drew B., Phaneuf S., Kirks A., Selman C., Gredilla R., Lezza A., Barja G., Leeuwenburgh C. (2002), Am. J. Physiol. Regul. Integr. Comp. Physiol., 284: R474-480.
6.Lee C.-K, Weindruch R., Prolla T.A. (2000), Nature Genetics, 25: 294-297
Key words:
aging rat, Real Time PCR, caloric restriction, mtDNA deletion
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