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Mitochondrial ROS and mtDNA fragments inside nuclear DNA as a main effector of ageing: the “cell aging regulation system”
high longevity. This makes them ideal to solve the
problem mentioned above. The lower mitROSp of
pigeons, canaries and parakeets, when compared to rats in
the first case and to mice in the second and third case,
strongly reinforces the MFRTA since it indicates that the
low mitROSp of long-lived animals occurs both in
comparisons between animals following Pearl´s law as
well as in those not following it. A high longevity is not a
simple consequence of a slow rate of living. It can be
obtained –as the bird case shows- together with high rates
of oxygen consumption and animal aerobic activity. High
longevity in the studied birds is associated with a low rate
of mitROSp both in absolute terms, and also as percentage
of mitochondrial oxygen consumption and thus of electron
flow at the electron transport chain (birds have a low
percent free radical leak, FRL).
3.2. The mitROSp site at the ETC important for longevity
It was been widely believed for decades that complex
III of the respiratory chain was the respiratory complex
responsible for ROS production in the ETC (the
mitochondrial electron transport chain) (13). Later it was
found, already working with freshly isolated and well
coupled functional mitochondria, that complex I also
produces ROS in heart or brain mitochondria isolated from
rats, mice, pigeons, canaries and parakeets (12, 14), which
was soon confirmed in rats by other laboratories (15, 16)
and soon became established knowledge in biochemistry
books.
A key experiment to detect complex I ROS production
was to measure mitROSp with succinate alone as well as
with succinate+rotenone. In the second situation the rate of
mitROSp acutely decreases because rotenone does not
allow the electrons to flow back to complex I from
succinate-complex II through reverse electron flow (17).
But the habitual procedure of adding succinate alone,
followed or not by antimycin A, and rarely using complex
I-linked substrates, led to the general but erroneous believe mtROS generation, it was logical to expect that this should
during decades that mitROS came mainly from complex
III-semiquinone.
In addition, we also found that the lower mtROS
generation rate observed in birds compared to mammals of
similar body size and weight-specific metabolic rate
occurred only at complex I (12, 14, 17), not at complex III.
This is most interesting since we found the same
afterwards in dietary restriction (DR) rat models (see
section 5). Concerning the precise site within complex I
where ROS are produced three generators have been
suggested, the flavin at the beginning of the electron path
within the complex, the FeS clusters of the hydrophilic
matrix domain, and the ubiquinone located in the
membrane domain. Various investigators have supported
the role of the flavin based on experiments with the
inhibitor diphenyliodonium, which strongly decreases
mitROSp. However, the site of action of
diphenyliodonium, at the beginning of the electron path,
also avoids electrons to reach the other two possible
generators, the various FeS clusters and the ubiquinone,
which therefore can not be discarded in those experiments.
@Real Academia Nacional de Farmacia. Spain
In other investigations, it was concluded that electron leak
to oxygen occurred between the ferricyanide reduction site
and the rotenone binding site of Complex I both in intact
mitochondria (12, 14, 17) as well as in submitochondrial
particles (18). Iron-sulphur clusters with a higher midpoint
potential than FeSN1a, which could be situated in the
electron path after the ferricyanide reduction site (15,18),
or the unstable semiquinone known to be present in the
membrane domain of Complex I and possibly functioning
in H+ pumping coupled to electron transport (19), could be
the complex I oxygen radical generators. However, many
different Complex I FeS clusters could be responsible for
complex I mitROSp because, under physiological
conditions: (a) their reduced and oxidized states will not be
present in equal concentrations; (b) interactions with many
different factors and surrounding macromolecules could
modify the final redox potential of the carriers in vivo; and
(c) the exact position of many FeS clusters in the Complex
I electron path is still unknown. Thus, the important aging-
related question whether flavin, FeS clusters or
ubisemiquinone, or a combination of these, are responsible
for the complex I ROS generation relevant for aging was
still unanswered (but see the proteomics ETC study
described at section 5, Ref. 53).
3.3. Oxidative damage to mtDNA and longevity
The location where mitochondrial DNA (mtDNA) is
situated is very close to the site of mtROS generation, the
ETC at the inner mitochondrial membrane. ROS
production also occurs at other cellular sites like
microsomes, peroxisomes or membrane-bound NADPH-
oxidases, and the rate of ROS generation at those sites can
substantially exceed in various situations that coming from
mitochondria. However, the ROS produced at
mitochondria can be still the most important ones for
longevity due to the presence of mtDNA within the
mitochondria but not at those other organelles or parts of
the cell. Since long-lived animal species have low rates of
have an effect on the steady-state level of oxidative
damage in their mtDNA. Therefore we decided to measure
the level of 8-oxodG (8-oxo-7,8-dihydro-
2’deoxyguanosine) in the heart and brain mitochondrial
and nuclear DNA of eight different mammalian species
differing by up to 13-fold in longevity. The results showed
that the level of 8-oxodG in the mtDNA of both organs is
negatively correlated with longevity (20). The longer the
longevity of a species, the smaller is its mtDNA oxidative
damage degree (Figure 1C). In contrast, the 8-oxodG level
in nuclear DNA (nDNA) did not correlate with longevity
in any organ even though mitochondrial and nuclear DNA
was measured in the same samples taken from the same
individual animals (20). Therefore, the different mitROSp
rates of the different species seem to have a direct impact
on mtDNA, and not on nDNA, concerning oxidative
damage. This makes sense since the site of ROS
generation at mitochondria is very close to mtDNA
whereas nDNA is situated far away from it inside the
nuclear compartment.
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