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The rate of mitROSp is measured in isolated
mitochondria in vitro due to the lack of reliable methods
for direct in vivo mitochondrial H2O2 production
determination. However, since the variations in the steady-
state levels of 8-oxodG in mtDNA closely reflect the
variations in the rate of mitROSp, both in comparative
(Figure 1B,C) and in dietary restriction (DR; section 5)
studies, suggests that the mitROSp in vitro measurements
were closely indicative of the situation in vivo. In addition, GSH-peroxidases, were applied to this problem, the results
the level of 8-oxodG in mtDNA was generally lower in the
heart and brain of three long-lived birds when compared to
two short-lived mammals of similar body size and specific
metabolic rate, in agreement with the superior longevity of
the birds, whereas again this was not the case for nDNA
(21). These investigations also showed that the intensity of
oxidative damage is several fold higher in mtDNA than in
nDNA in the heart and brain of all the 11 different species
of mammals and birds studied (20, 21), which is again
consistent with the close proximity between mtDNA and
the sites of mtROS generation. Studies on the longest-
lived metazoan, the bivalve A. islandica have also shown
increases with age in DNA oxidation and lower DNA
oxidative damage than in other bivalve populations with
much shorter (6 fold lower) longevities (22).
3.4. Increasing antioxidants does not change longevity
Initial studies about MFRTA were mainly focused on
antioxidants because they were easier to measure, and
because sensitive enough techniques to assay mitROSp in
different species were generally not available at that time
mainly due to the generalized use of spectrophotometry
instead of fluorometry. Most of the investigations on the
effect of adding dietary antioxidants to the diet were
performed during the 1970´s and 80´s. The general result
was that antioxidants did not increase (maximum)
longevity (23, 24). In some experiments they increased
only mean longevity (23-25). Interestingly, this tended to
occur when the (maximum) longevity of the control
rodents was short, usually less than 3 years. This suggests
that antioxidants, when the husbandry conditions are
suboptimal, could protect from causes of early death, and
thus they can make more rectangular the survival curve.
This is what happened in humans during the XXth century
in many developing western countries when mean life
expectancy increased from around 40 to 80 years without
decreasing in aging rate. That is why now the old are so
abundant in western populations. Antioxidants, in such
cases above, were bringing back towards optimum the
diminished survival of the controls reared under
suboptimum environmental conditions, which is
interesting but not the goal of gerontology. Ironically, the
poorer the survival curve of the controls, the largest is the
chance of obtaining a positive result in terms of mean
longevity. A most inappropriate stimulus to not well done
research indeed. Whereas, the better one performs the
experiment, the less chances one has of obtaining an
increase in mean longevity. In summary, like in the
comparative inter-specific studies described above,
antioxidants clearly can not be among the factors
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Gustavo Barja de Quiroga
responsible for low aging rates and high (maximum)
longevities.
When the techniques to obtain transgenic or knockout
mice with increased or lack of expression of genes
codifying for antioxidant enzymes like SODs, catalase, or
were (obviously) similarly disappointing (26, 27). The
antioxidant enzyme activities increased through
modification of gene expression, like the non enzymatic
dietary antioxidants added to the diet, did not slow aging.
Independently of the way in which the antioxidants were
manipulated, dietary or genetic, the result was the same: a
lack of effect of antioxidants on mammalian longevity.
This has been interpreted by some (27) but not other (28)
authors as the “death” of the MFRTA (27), but such
conclusion (27) did not take into account that what
correlates with longevity in the right sense is not the level
of the antioxidants, but the mitROSp rate and the fatty acid
unsaturation degree of the cellular membranes (23, 29).
Studies in simpler organisms like the fungus
Podospora anserina have provided strong evidence for a
role of mitROSp in senescence including DR-effects, and
strains of this fungus deprived of mitROSp are converted
to "eternal" non aging organisms (30). Recent experiments
in 29 different longevity mutants, and 26 different
environmental situations and drugs that increase (DR, PR,
MetR, rapamycin, starvation, and others) or decrease
(including 10 or 20 mg/l glucose medium, the 10µM
amyloid-binding compound ThT, 1mM 4,4´-
diaminodiphenylsulphone DDS, 10 mM N-acetyl cysteine)
longevity used a new fluorescent probe claimed to estimate
mitROSp (“free radical production and metabolic activity
at the single-mitochondrion level”, Ref. 31) for the first
time in vivo. The results of these studies, performed in the
nematode worm C. elegans, were extremely interesting. In
all the 55 cases lower "mitROSp" (mitoflash) was always
observed in log-lived animals (31). These results were
quickly and heavily criticized in the same journal (Nature)
by 27 other researchers from different fields related or not
gerontology. The critics claimed that what the mitoflash
technique estimated was not mitROSp (superoxide radical)
because changes in fluorescence of the cpYFP used (the
sensor circularly permuted yellow fluorescent protein in
the matrix) “are due to alterations in pH, not superoxide".
Thus, the critic was exclusively methodological, and did
not consider what could be the biological significance of
Shen et al. results (31). The reply of the authors of the C.
elegans mitoflash study was also published in Nature
together with the critic. The authors of the mitoflash study
have continued publishing further investigations since
2014 using the same technique, leading to around a dozen
further publications, some in mouse muscle, and in some
studies they tried to ascertain if H+ indeed interfered or not
with their mitoflashes. The issue does not seem still
resolved today. But if the critics are correct, why then low
mitoflash (as claimed partial estimator of "mROSp") was
always observed in long-lived animals, in full agreement
with hundreds of published mitROSp-longevity studies
@Real Academia Nacional de Farmacia. Spain