Báo cáo Y học: The reductive hotspot hypothesis of mammalian aging Membrane metabolism magnifies mutant mitochondrial mischief pptx

MINIREVIEW
The reductive hotspot hypothesis of mammalian aging
Membrane metabolism magnifies mutant mitochondrial mischief
Aubrey D. N. J. de Grey
Department of Genetics, University of Cambridge, UK
A severe challenge to the idea that mitochondrial DNA
mutations play a major role in the aging process in mammals
is that clear loss-of-function mutations accumulate only to
very low levels (under 1% of total) in almost any tissue, even
by very old age. Their accumulation is punctate: some cells
become nearly devoid of wild-type mitochondrial DNA and
exhibit no activity for the partly mitochondrially encoded
enzyme cytochrome c oxidase. Such cells accumulate in
number with aging, suggesting that they survive indefinitely,
which is itself paradoxical. The reductive hotspot hypothesis
suggests that these cells adjust their metabolism to use
plasma membrane electron transport as a substitute for the
mitochondrial electron transport chain in the reoxidation of
reduced dinucleotides, and that, like mitochondrial electron
transport, this process is imperfect and generates superoxide
as a side-effect. This superoxide, generated on the outside of
the cell, can potentially initiate classical free radical chem-
istry including lipid peroxidation chain reactions in circula-
ting material such as lipoproteins. These, in turn, can be toxic
to mitochondrially nonmutant cells that import them to
satisfy their cholesterol requirements. Thus, the relatively
few cells that have lost oxidative phosphorylation capacity
may be toxic to the rest of the body. In this minireview,
recent results relevant to this hypothesis are surveyed and
approaches to intervening in the proposed process are dis-
cussed.

Keywords: aging; mitochondrial mutations; plasma
membrane redox; extracellular superoxide; lipoproteins.
INTRODUCTION
A large and compelling body of evidence has been
assembled over the past 30 years in support of Harman’s
1972 proposal [1] that oxidative damage to mitochondria,
resulting from the adventitious production of superoxide by
the respiratory chain, is a major determinant of the rate of
aging. The most direct such evidence is the finding that
mitochondrial superoxide production rates (measured as a
proportion of respiration rate) correlate with rates of aging,
when comparing either closely or distantly related species
[2–5] or when calorically restricted animals are compared to
ad libitum-fed animals [6]. Moreover, the mitochondrial
form of superoxide dismutase is the only one whose deletion
in mice is lethal [7]; homozygous knockouts of the cytosolic
and extracellular forms show only mild phenotypes and no
dramatic shortening of lifespan [8,9].
The role of mitochondria as mediators of oxidative
damage leading to aging is made especially plausible by their
possession of their own genome (the mitochondrial DNA,
or mtDNA). The mtDNA encodes proteins essential for
aerobic respiration and its proximity to the cell’s major
source of free radicals renders it highly susceptible to
mutagenic insults. Furthermore, it is the only component of
mitochondria in which damage can accumulate, because
their protein and lipid constituents are periodically rejuven-
ated by the division of mitochondria that occurs in all cells
(even postmitotic ones, in which it is balanced by mito-
chondrial autophagocytosis [10]). Mitochondrial biogenesis

entails the incorporation into mitochondria of pristine,
undamaged lipids and proteins, thus diluting any damage
that may be present.
Although mtDNA mutations can theoretically accumu-
late even in the face of mitochondrial turnover, one would
not expect them to do so: a more natural presumption
would be that mitochondria housing mutant mtDNA
would be preferentially eliminated by turnover, resulting
in a low and nonincreasing level of mutant mtDNA. Indeed,
Comfort pointed this out as long ago as 1974 [11]. However,
it is now clear that, paradoxical though it may seem, the
opposite happens: loss-of-function mtDNA mutations,
especially large deletions, clonally expand in many cell
types at the expense of wild-type genomes, resulting in cells
that possess no oxidative phosphorylation (OXPHOS)
function as measured by histochemistry [12–14]. This may
occur via diminished autophagocytosis of mitochondria
that are not performing OXPHOS and thereby generating
less superoxide [15], as it is now clear that, contrary to the
once widely accepted vicious cycle theory [16], the absence
of all 13 mtDNA-encoded proteins, which is the result of
any large deletion as tRNA genes are always affected,
precludes the assembly of Complexes I [17] and III [18] and
thus prevents ubisemiquinone formation.
Correspondence to A. D. N. J. de Grey, Department of Genetics,
University of Cambridge, Downing Street, Cambridge CB2 3EH,
Fax: + 44 1223 333992, Tel.: + 44 1223 333963,
E-mail:
Abbreviations: mtDNA, mitochondrial DNA; OXPHOS, oxidative
phosphorylation; PMRS, plasma membrane redox system; RHH,

reductive hotspot hypothesis; EC-SOD, extracellular superoxide
dismutase; COX, cytochrome c oxidase; LDL, low-density
lipoprotein.
(Received 28 November 2001, revised 4 February 2002, accepted
6 February 2002)
Eur. J. Biochem. 269, 2003–2009 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.02868.x
HOW ABUNDANT ARE TRUE
LOSS-OF-FUNCTION mtDNA
MUTATIONS?
On closer inspection, however, the selective advantage
enjoyed by dysfunctional mtDNA constitutes a challenge to
the ÔmtDNA theory of agingÕ, rather than a reinforcement of
it. Except in the substantia nigra [19], under 1% of cells
become OXPHOS-negative even by very old age [20,21].
Other cells appear mitochondrially healthy. Any OXPHOS-
positive cell that also harbours high levels of dysfunctional
mtDNA should rapidly become OXPHOS-negative as a
result of selection for the mutant species; the slow rate of
increase with aging in the number of OXPHOS-negative
cells thus implies that few cells are in this highly hetero-
plasmic state.
In recent years, the above logic has been challenged by
reports of very high levels of deletion-bearing mtDNA in
tissues of older people [22–24]. The values reported were so
high as to seem inconsistent with the retention of essentially
undiminished bioenergetic capacity [25], but they ostensibly
supported the Ôtip of the icebergÕ hypothesis [26] that the low
levels of deletion-bearing mtDNA seen in tissue homogen-
ates were a result of the technical difficulty of detecting all
possible deletions by PCR. More recent work, however, has

cast doubt on this interpretation. It seems quite likely that
the bulk of mutations detected in the earlier studies were in
fact partial duplications rather than deletions; these alter-
natives can be distinguished by designing custom primers, a
technique that has recently shown that duplications are
indeed present in tissue [27,28]. Another possibility is that
the deleted mtDNA found in the earlier studies [23,24] was
in the process of being degraded: the finding [29] that most
of the oxidized bases in mtDNA are on fragments, rather
than full-length molecules, suggests that the mitochondrion
may use wholesale destruction of damaged mtDNA mol-
ecules (coupled with replication of undamaged ones) as a
repair mechanism.
Partial duplications deserve close attention, as they may
be much more abundant than deletions. However, no
evidence yet exists to suggest that they are phenotypically
significant except in very rare cases. A typical duplication
should give rise to transcripts for all 37 mtDNA-encoded
gene products, so the only route by which it could be
dysfunctional is if the altered stoichiometry of those
products impairs translation or assembly of the respirat-
ory chain complexes. That this seems not to be so is
indicated by the much lower levels of cytochrome c
oxidase (COX)-negative cells than cells with predomin-
antly duplicated mtDNA (though a similar comparison
for the other three partly mtDNA-encoded enzymes
would be needed in order to address this matter
thoroughly). Hence, perhaps duplications are more com-
mon than deletions partly because they are harmless, so
that evolution has not selected for mechanisms to

suppress their occurrence to the same extent as for
deletions. The duplicated region often includes the origin
of mtDNA heavy strand replication, and both such
origins are functional in such molecules [30]; this may
drive clonal expansion of the duplication relative to wild-
type by replicative advantage, as opposed to slower
autophagocytosis. (There is evidence for a similar multi-
plicity of mechanisms of expansion in suppressive petite
yeast [31].) Similarly, no evidence has yet come to light for
functional impairment of mtDNA carrying point muta-
tions in the noncoding D-loop region; some of these
accumulate with age, possibly also by accelerating repli-
cation [32].
THEPLASMAMEMBRANEREDOX
SYSTEM: LOCAL GOOD, GLOBAL
HARM?
If the only effect of mtDNA mutation is to generate a very
small number of cells lacking OXPHOS function, how can
damage to mtDNA matter at the organismal level (i.e. drive
aging)? Any such connection would seem to require that
those few cells be actively toxic, rather than just bioener-
getically dysfunctional. A hypothesis along such lines was
put forward by the present author recently [33,34] and is
summarized here (see Fig. 1).
OXPHOS directly maintains two aspects of cellular
homeostasis: the ATP/ADP ratio and the NAD
+
/NADH
ratio. Yeast cells can survive without OXPHOS (as petite
strains) because they can maintain ATP supply using

glycolysis and also keep a stable NAD
+
/NADH ratio by
reduction of the resulting pyruvate. Mammalian cells,
however, die when deprived of their mtDNA unless
additional, exogenous pyruvate is provided in the medium
Fig. 1. Overview of the reductive hotspot hypothesis (modified with
permission from [34]). Rare cells that have been taken over by mutant
mitochondria reduce LDL-bound haemin via superoxide; LDL per-
oxidation results; cells which import such LDL may suffer increased
oxidative stress, especially in occasional cases of damage to lysosomes.
The age-related rise in systemic oxidative stress and damage is thus
proposed to originate mainly from the accumulation of mitochondri-
ally mutant cells.
2004 A. D. N. J. de Grey (Eur. J. Biochem. 269) Ó FEBS 2002
[35]. This indicates that, though OXPHOS is still dispen-
sable for maintaining ATP supply, mammalian cells cannot
emulate yeast’s ability to Ôbalance the booksÕ with regard to
redox state by reducing glycolysis-derived pyruvate to
lactate and exporting it; an additional electron sink is
needed.
Extracellular pyruvate is virtually absent in vivo,
however, so for OXPHOS-negative cells to survive
indefinitely (which they evidently do, or else they should
not accumulate with age) they must use some other
electron acceptor. Molecular oxygen may be the only
acceptor available in sufficient abundance. Importantly, it
was shown that ferricyanide could substitute for pyruvate
in supporting growth of mtDNA-less (q°) mammalian
cells [36]; as ferricyanide cannot enter the cell and NADH

cannot exit it, this shows that a system exists in the
plasma membrane that can oxidize cytosolic NADH and
transfer the resulting electrons to an extracellular acceptor.
Such a system has long been known – in fact, see [37] for
a wide-ranging review of early work – though it is still
only poorly characterized. It is termed the plasma
membrane redox system (PMRS).
In summary, it is therefore theoretically possible that
OXPHOS-negative cells could survive by reducing oxygen
at the plasma membrane rather than at the mitochondrial
inner membrane. The rate at which they do so may be
extremely high, as histochemical evidence [13,14,20] of
markedly elevated succinate dehydrogenase, even if nor-
malized to mtDNA content [38], suggests that such cells do
not rely solely on glycolysis but also maintain an active TCA
cycle, which entails a far greater rate of reduction (and hence
reoxidation) of NAD. This may be possible only by
reversing the usual direction of the malate/aspartate and
glycerophosphate shuttles; the former operates close to
thermodynamic equilibrium [39] but the latter may require
substantial shifts in cellular state in order to be reversed.
(The possibility that electrons from Complex II are fed to
cytosolic NAD by a route other than coenzyme Q and the
glycerophosphate shuttle must also be kept in mind,
however.)
The possible drawback of this system for the organism is
analogous to the drawback of OXPHOS itself: namely, that
in certain circumstances the oxygen used as a terminal
electron acceptor may be reduced not to water but to
superoxide. (It is this aspect of the proposal that has given it

the name Ôreductive hotspot hypothesisÕ, abbreviated RHH:
as superoxide is a reductant, such cells constitute a punctate
source of reductive stress.) Because this is proposed to occur
on the cell surface, it is potentially a threat to oxidisable
circulating material such as low-density lipoprotein (LDL)
particles, especially if they are in contact with redox-active
transition metals (as they sometimes may be [40]) that can
convert the reductive stress of superoxide to oxidative stress
from hydroxyl and alkoxyl radicals. LDL oxidation may
play a key role in atherosclerosis [41]; more generally,
however, slightly oxidized LDL is readily imported by most
cell types in the course of meeting their cholesterol needs
[42], so it may be a source of oxidative stress in cells that
retain OXPHOS competence (i.e. the vast majority of our
cells). The mechanism of release of cholesterol from the
vacuolar apparatus after endocytosis is still obscure [43], but
the presence of oxysterols in that compartment may inhibit
the release of unoxidized cholesterol [44] or even stimulate
lysosomal rupture [45], with potentially severe consequences
for the cell.
RECENT DATA PERTINENT TO RHH
The attractiveness of such an elaborate hypothesis is
necessarily dependent on persuasive evidence. Initially, only
rather indirect evidence was available, such as the high
succinate dehydrogenase activity of COX-negative muscle
fibre segments (which might be explained as futile compen-
sation for OXPHOS failure) and the high rate of superoxide
production by cells exposed to extracellular NADH [46]
(which is nonphysiological). Recent reports have substan-
tially enhanced the array of evidence that something like the

reductive hotspot mechanism is present in vivo and may be
involved in aging.
Efforts to dissect the PMRS have been relatively
successful for the cytosolic-side, NADH-oxidizing compo-
nents but less so for the downstream, cell-surface ones.
Cytochrome b
5
reductase and DT-diaphorase, and prob-
ably at least one other enzyme, transfer electrons from
NADH to plasma membrane coenzyme Q. The involve-
ment of cytochrome b
5
reductase (but not, interestingly, of
cytochrome b
5
) [47] opens the possibility of one-electron
redox processes being involved, which make it more likely
that superoxide could be formed.
The group of Morre
´
have cloned an enzyme that may be
the terminal electron transfer protein in the PMRS of
tumour cells, but is absent from normal cells [48]. It is
detectable in the serum of cancer patients. Moreover, a
constitutive enzyme with the same activity is present in
serum of older healthy individuals at higher levels than in
young people [49]. As further evidence that a redox chain
exists linking cytosolic NADH to extracellular superoxide,
Berridge & Tan have shown [50] that cultured cells can
generate substantial extracellular superoxide when oxidizing

cytosolic, rather than just extracellular, NADH.
In vivo assays for production of extracellular superoxide
and hydrogen peroxide have revealed that it is markedly
elevated in skeletal muscle by acute exercise [51] and in heart
by ischaemia/reperfusion [52]. The significance of this is that
both treatments would be predicted to cause depletion of
oxygen at the mitochondrial respiratory chain but less so at
the cell surface, so a PMRS-based respiration mechanism
may be stimulated. Moreover, this would imply that the
PMRS is already present in such cells, rather than being
induced by mtDNA mutation accumulation; indeed, PMRS
activity is remarkably ubiquitous, found in all cell types so
far examined [53].
Finally, evidence has been provided that links the PMRS
to aging. Desai et al. [54] found that caloric restriction,
which extends both mean and maximum lifespan of rodents,
causes a threefold reduction in activity of Complex I in
muscle but no change in Complex II activity. Because the
TCA cycle provides nearly all Complex I’s substrate and all
of Complex II’s, they would be expected to respond
similarly to any long-term intervention. That they do not
suggests that much of the NADH produced by the TCA
cycle may be diverted out of the mitochondrion
1
(by a
reversed malate/aspartate shuttle) and cellular redox stabil-
ity maintained by the PMRS [55]. This is a plausible
mechanism for the life-extending effects of caloric restric-
tion, because Complex I is the mitochondrion’s main
Ó FEBS 2002 The reductive hotspot hypothesis (Eur. J. Biochem. 269) 2005

superoxide generator in physiological conditions [56], so
reducing its activity should reduce free radical production.
That might be of little benefit if superoxide production
occurred at the cell surface instead, as RHH proposes, but
the presence of a functional Complex III and IV gives a very
different situation than is proposed for mitochondrially
mutant cells: in particular, the glycerophosphate shuttle
need not be reversed. This may allow the PMRS to be
elevated ÔcleanlyÕ, without concomitant superoxide produc-
tion (Fig. 2).
REMAINING AVAILABLE TESTS:
BIOMEDICAL SIGNIFICANCE
A very direct challenge to RHH is the lack of an acceleration
of aging in mice homozygous for a knockout of extracellular
superoxide dismutase (EC-SOD) [9]. This might be because
oxygen is not the principal electron acceptor for the PMRS
of OXPHOS-less cells, but alternative acceptors are not
apparent. Another possibility is that the level of EC-SOD in
muscle, which is the tissue most implicated in RHH on
account of its abundance in the body, may simply be too low
to metabolize much of the superoxide produced so focally
by such cells [57]. Muscle-specific overexpression of
EC-SOD in mice could shed light on this issue: RHH
predicts that this would diminish steady-state levels of
oxidation of circulating LDL and extend lifespan.
Some other direct tests of RHH also involve interventions
that RHH predicts would be life-extending, unless they had
harmful side-effects. Inhibitors of the PMRS are an
attractive option, as they should prevent the formation of
extracellular superoxide. However, the PMRS’s extreme

ubiquity suggests that it may play an important, unidenti-
fied role in cellular stability, perhaps as a redox buffer [58];
indiscriminate inhibition might therefore be toxic. More-
over, OXPHOS-negative muscle fibre segments that were
prevented from using the PMRS to survive might atrophy
and potentially kill the entire fibre, risking severe sarcopenia
along the lines suggested by Aiken [59].
If RHH is broadly correct, life-extending interventions can
also be conceived that act to restore or maintain OXPHOS
competence despite the inevitable occurrence of mtDNA
mutations. Such interventions would only be Ôone-sidedÕ tests
of RHH, their inefficacy would falsify RHH; but their
efficacy would be consistent with other mechanisms whereby
OXPHOS dysfunction might lead to aging. However, the
medical relevance of such interventions merits their careful
analysis, so they are the topic of the remainder of this section.
One possibility is selectively to inhibit the biogenesis of
mitochondria that are OXPHOS-negative. This is a partic-
ularly promising approach in muscle because, if carried out
gradually enough, the OXPHOS-positive regions of the
fibre on either side of the affected segment could potentially
repopulate it with wild-type mitochondria, leading to a
shrinkage and eventual disappearance of the defect without
any atrophy or death of the fibre. Approaches to achieving
this include inhibition of mitochondrial protein import,
which may already be somewhat hampered by the reduced
proton gradient of mutant mitochondria [60] so may be
adequately selective.
Such approaches may be insufficiently ambitious
2

, how-
ever. Absolute avoidance of any deleterious effects of
mtDNA mutations could be achieved by completing the job
that evolution has left unfinished; engineering transgenic
nuclear copies of the 13 protein-coding genes of the
mtDNA, suitably modified so that their products still have
the correct amino acid sequence and are imported into
mitochondria for assembly into the respiratory chain. This
strategy (known as allotopic expression) was first achieved
in yeast, with full phenotypic rescue of a mitochondrial
deletion for the corresponding gene, as long ago as 1988
[61]; further progress was slow for many years thereafter but
has greatly accelerated recently [62,63], including success in
mammalian cells by two groups [64,65]. The recent cloning
of three of the relevant genes from Chlamydomonas,in
which they are nuclear-coded, has given further insight into
how to modify such genes so that their encoded proteins’
hydrophobicity does not prohibit import [66].
Variations on this theme are also worthy of considera-
tion. Import of mRNA rather than protein might be
sufficient if translation can be induced after import (and if
all mitochondrial tRNAs and rRNAs are also imported);
import of short RNAs into mammalian cells has been
engineered [67]. However, no case of mRNA being
imported into mitochondria has been discovered in any
Fig. 2. Proposed response of cells to reduction or elimination of com-
plex I activity. Caloric restriction is proposed to cause reversal of the
malate/aspartate shuttle, which entails only modest shifts of redox
state or membrane potential so may not promote plasma membrane
superoxide production. Elimination of complexes III and IV, by

contrast, prevents operation of the TCA cycle unless the glycero-
phosphate shuttle is also reversed, a thermodynamically more difficult
task; the associated changes in cytosolic redox state may promote
plasma membrane superoxide production.
2006 A. D. N. J. de Grey (Eur. J. Biochem. 269) Ó FEBS 2002
organism, so this may prove very challenging. An ingeni-
ous alternative is to introduce genes from other organisms
whose products perform the electron transport functions of
the respiratory chain without pumping protons; these are
already nuclear-coded, so their introduction into mamma-
lian cells is comparatively straightforward. Indeed, yeast
NDI1hasbeenexpressedinmammaliancellsandshown
to complement Complex I inactivation [68]. If it were
coexpressed with the alternative oxidase, which in many
organisms transfers electrons from ubiquinol to oxygen,
the endogenous electron transport chain would be entirely
sidestepped. This would clearly be deleterious if carried out
constitutively, as it would prevent ATP synthesis by
OXPHOS, but if somehow induced only when a cell
became OXPHOS-negative, or if the enzymes were chosen
or modified so as to have a somewhat lower affinity for
their substrate than the corresponding proton-pumping
enzymes, such that they did not compete with them, then
RHH would predict that the toxicity of OXPHOS-negative
cells (and hence of mtDNA mutations in aging) would be
prevented, as those cells’ internal redox homeostasis would
remain intact and elevation of the PMRS should not occur.
CONCLUSION
Though it may at first seem unattractively elaborate, the
reductive hotspot hypothesis of mammalian aging is an

extension of the long-standing mitochondrial theory that,
unlike many of its predecessors, remains strikingly consis-
tent with available evidence. It is not the only hypothesis
with that quality, however, and experiments to test it are
merited. The increasing recognition that earlier, simpler
models for mitochondrion-driven aging are inadequate has
already stimulated much relevant work, which has been
briefly surveyed here and in the accompanying minireviews
by Brunk & Terman and by McKenzie et al.;thistrend
seems set to continue and to bring light to what is widely
considered a primary mechanism underlying mammalian
aging.
REFERENCES
1. Harman, D. (1972) The biologic clock: the mitochondria? J. Am.
Geriatr. Soc. 20, 145–147.
2. Sohal, R.S., Arnold, L.A. & Sohal, B.H. (1990) Age-related
changes in antioxidant enzymes and prooxidant generation in
tissues of the rat with special reference to parameters in two insect
species. Free Radic. Biol. Med. 9, 495–500.
3. Barja, G., Cadenas, S., Rojas, C., Pe
´
rez-Campo, R. & Lo
´
pez-
Torres, M. (1994) Low mitochondrial free radical production per
unit O
2
consumption can explain the simultaneous presence of
high longevity and high aerobic metabolic rate in birds. Free
Radic. Res. 21, 317–327.

4. Ku, H.H. & Sohal, R.S. (1993) Comparison of mitochondrial pro-
oxidant generation and anti-oxidant defenses between rat and
pigeon: possible basis of variation in longevity and metabolic
potential. Mech. Ageing Dev. 72, 67–76.
5. Sohal, R.S., Ku, H.H. & Agarwal, S. (1993) Biochemical corre-
lates of longevity in two closely related rodent species. Biochem.
Biophys. Res. Commun. 196, 7–11.
6. Gredilla, R., Sanz, A., Lo
´
pez-Torres, M. & Barja, G. (2001)
Caloric restriction decreases mitochondrial free radical generation
at complex I and lowers oxidative damage to mitochondrial DNA
in the rat heart. FASEB J. 15, 1589–1591.
7. Li, Y.B., Huang, T.T., Carlson, E.J., Melov, S., Ursell, P.C.,
Olson, J.L., Noble, L.J., Yoshimura, M.P., Berger, C., Chan,
P.H., Wallace, D.C. & Epstein, C.J. (1995) Dilated cardiomyo-
pathy and neonatal lethality in mutant mice lacking manganese
superoxide dismutase. Nat. Genet. 11, 376–381.
8. Re
´
aume, A.G., Elliott, J.L., Hoffman, E.K., Kowall, N.W.,
Ferrante, R.J., Siwek, D.F., Wilcox, H.M., Flood, D.G., Beal,
M.F., Brown, R.H., Scott, R.W. & Snider, W.D. (1996) Motor
neurons in Cu/Zn superoxide dismutase-deficient mice develop
normally but exhibit enhanced cell death after axonal injury. Nat.
Genet. 13, 43–47.
9. Carlsson, L.M., Jonsson, J., Edlund, T. & Marklund, S.L. (1995)
Mice lacking extracellular superoxide dismutase are more sensitive
to hyperoxia. Proc.NatlAcad.Sci.USA92, 6264–6268.
10. Menzies,R.A.&Gold,P.H.(1971)Theturnoverofmitochondria

in a variety of tissues of young adult and aged rats. J. Biol. Chem.
246, 2425–2429.
11. Comfort, A. (1974) The position of aging studies. Mech. Ageing
Dev. 3, 1–31.
12. Mu
¨
ller-Ho
¨
cker, J., Seibel, P., Schneiderbanger, K. & Kadenbach,
B. (1993) Different in situ hybridization patterns of mitochondrial
DNA in cytochrome c oxidase-deficient extraocular muscle fibres
in the elderly. Virchows Arch. 422, 7–15.
13. Mu
¨
ller-Ho
¨
cker, J. (1990) Cytochrome c oxidase deficient fibres in
the limb muscle and diaphragm of man without muscular disease:
an age-related alteration. J. Neurol. Sci. 100, 14–21.
14. Lee, C.M., Lopez, M.E., Weindruch, R. & Aiken, J.M. (1998)
Association of age-related mitochondrial abnormalities with ske-
letal muscle fiber atrophy. Free Radic. Biol. Med. 25, 964–972.
15. de Grey, A.D.N.J. (1997) A proposed refinement of the mito-
chondrial free radical theory of aging. BioEssays 19, 161–166.
16. Bandy, B. & Davison, A.J. (1990) Mitochondrial mutations may
increase oxidative stress: implications for carcinogenesis and
aging? Free Radic. Biol. Med. 8, 523–539.
17. Bai, Y. & Attardi, G. (1998) The mtDNA-encoded ND6 subunit
of mitochondrial NADH dehydrogenase is essential for the
assembly of the membrane arm and the respiratory function of the

enzyme. EMBO J. 17, 4848–4858.
18. Sen, K. & Beattie, D.S. (1986) Cytochrome b is necessary for the
effective processing of core protein I and the iron-sulfur protein of
complex III in the mitochondria. Arch. Biochem. Biophys. 251,
239–249.
19. Itoh, K., Weis, S., Mehraein, P., Mu
¨
ller-Ho
¨
cker, J. (1996) Cyto-
chrome c oxidase defects of the human substantia nigra in normal
aging. Neurobiol. Aging 17, 843–848.
20. Mu
¨
ller-Ho
¨
cker, J. (1989) Cytochrome c oxidase deficient cardio-
myocytes in the human heart – an age-related phenomenon. A
histochemical ultracytochemical study. Am. J. Pathol. 134, 1167–
1173.
21. Cooper, J.M., Mann, V.M. & Schapira, A.H.V. (1992) Analyses
of mitochondrial respiratory chain function and mitochondrial
DNA deletion in human skeletal muscle: effect of ageing.
J. Neurol. Sci. 113, 91–98.
22. Nagley, P. & Wei, Y.H. (1998) Ageing and mammalian mito-
chondrial genetics. Trends Genet. 14, 513–517.
23. Hayakawa, M., Katsumata, K., Yoneda, M., Tanaka, M.,
Sugiyama, S. & Ozawa, T. (1996) Age-related extensive frag-
mentation of mitochondrial DNA into minicircles. Biochem.
Biophys. Res. Commun. 226, 369–377.

24. Kovalenko, S.A., Kopsidas, G., Kelso, J.M. & Linnane, A.W.
(1997) Deltoid human muscle mtDNA is extensively rearranged in
old age subjects. Biochem. Biophys. Res. Commun. 232, 147–152.
25. Barrientos, A., Casademont, J., Ro
¨
tig, A., Miro
´
, O., Urbano,
Ma
´
rquez, A., Rustin, P. & Cardellach, F. (1996) Absence of
relationship between the level of electron transport chain activities
and aging in human skeletal muscle. Biochem. Biophys. Res.
Commun. 229, 536–539.
Ó FEBS 2002 The reductive hotspot hypothesis (Eur. J. Biochem. 269) 2007
26. Cortopassi, G.A. & Arnheim, N. (1990) Detection of a specific
mitochondrial DNA deletion in tissues of older humans. Nucleic
Acids Res. 18, 6927–6933.
27. Bodyak, N.D., Nekhaeva, E., Wei, J.Y. & Khrapko, K. (2001)
Quantification and sequencing of somatic deleted mtDNA in
single cells: evidence for partially duplicated mtDNA in aged
human tissues. Hum. Mol. Genet. 10, 17–24.
28. Kajander, O.A., Rovio, A.T., Majamaa, K., Poulton, J., Spel-
brink, J.N., Holt, I.J., Karhunen, P.J. & Jacobs, H.T. (2000)
Human mtDNA sublimons resemble rearranged mitochondrial
genomes found in pathological states. Hum. Mol. Genet. 9, 2821–
3285.
29. Suter, M. & Richter, C. (1999) Fragmented mitochondrial DNA is
the predominant carrier of oxidized DNA bases. Biochemistry 38,
459–464.

30. Umeda,S.,Tang,Y.,Okamoto,M.,Hamasaki,N.,Schon,E.A.
& Kang, D. (2001) Both heavy strand replication origins are active
in partially duplicated human mitochondrial DNAs. Biochem.
Biophys. Res. Commun. 286, 681–687.
31. Chambers, P. & Gingold, E. (1986) A direct study of the relative
synthesis of petite and grande mitochondrial DNA in zygotes
from crosses involving suppressive petite mutants of Sacchar-
omyces cerevisiae. Curr. Genet. 10, 565–571.
32. Wang, Y., Michikawa, Y., Mallidis, C., Bai, Y., Woodhouse, L.,
Yarasheski, K.E., Miller, C.A., Askanas, V., Engel, W.K., Bhasin,
S. & Attardi, G. (2001) Muscle-specific mutations accumulate with
aging in critical human mtDNA control sites for replication. Proc.
Natl Acad. Sci. USA 98, 4022–4027.
33. de Grey, A.D.N.J. (1998) A mechanism proposed to explain
the rise in oxidative stress during ageing. J. Anti-Aging Med. 1,
53–66.
34. de Grey, A.D.N.J. (2000) The reductive hotspot hypothesis: an
update. Arch. Biochem. Biophys. 373, 295–301.
35. King, M.P. & Attardi, G. (1989) Human cells lacking mtDNA:
repopulation with exogenous mitochondria by complementation.
Science 246, 500–503.
36. Larm, J.A., Vaillant, F., Linnane, A.W. & Lawen, A. (1994)
Up-regulation of the plasma membrane oxidoreductase as a pre-
requisite for the viability of human Namalwa q
0
cells. J. Biol.
Chem. 269, 30097–30100.
37. Crane,F.L.,Sun,I.L.,Clark,M.G.,Grebing,C.&Lo
¨
w, H. (1985)

Transplasma-membrane redox systems in growth and develop-
ment. Biochim. Biophys. Acta 811, 233–264.
38. Moraes, C.T., Ricci, E., Petruzzella, V., Shanske, S., DiMauro, S.,
Schon, E.A. & Bonilla, E. (1992) Molecular analysis of the muscle
pathology associated with mitochondrial DNA deletions. Nat.
Genet. 1, 359–367.
39. Bremer, J. & Davis, E.J. (1975) Studies on the active transfer of
reducing equivalents into mitochondria via the malate-aspartate
shuttle. Biochim. Biophys. Acta 376, 387–397.
40. Miller, Y.I. & Shaklai, N. (1999) Kinetics of hemin distribution in
plasma reveals its role in lipoprotein oxidation. Biochim. Biophys.
Acta 1454, 153–164.
41. Steinberg, D. (1997) Low density lipoprotein oxidation and its
pathobiological significance. J. Biol. Chem. 272, 20963–20966.
42. Lyons, T.J., Li, W., Wells-Knecht, M.C. & Jokl, R. (1994)
Toxicity of mildly modified low-density lipoproteins to cultured
retinal capillary endothelial cells and pericytes. Diabetes 43, 1090–
1095.
43. Kobayashi, T., Beuchat, M.H., Lindsay, M., Frias, S., Palmiter,
R.D., Sakuraba, H., Parton, R.G. & Gruenberg, J. (1999) Late
endosomal membranes rich in lysobisphosphatidic acid regulate
cholesterol transport. Nat. Cell Biol. 1, 113–118.
44. Gaus, K., Dean, R.T., Kritharides, L. & Jessup, W. (2001)
Inhibition of cholesterol efflux by 7-ketocholesterol: comparison
between cells, plasma membrane vesicles, and liposomes as cho-
lesterol donors. Biochemistry 40, 13002–13014.
45. Yuan, X.M., Li, W., Brunk, U.T., Dalen, H., Chang, Y.H. &
Sevanian, A. (2000) Lysosomal destabilization during macrophage
damage induced by cholesterol oxidation products. Free Radic.
Biol. Med. 28, 208–218.

46. O’Donnell, V.B. & Azzi, A. (1996) High rates of extracellular
superoxide generation by cultured human fibroblasts: involvement
of a lipid-metabolizing enzyme. Biochem. J. 318, 805–812.
47. Villalba, J.M., Navarro, F., Go
´
mez-Dı
´
az, C., Arroyo, A., Bello,
R.I. & Navas, P. (1997) Role of cytochrome b5 reductase on the
antioxidant function of coenzyme Q in the plasma membrane.
Mol. Aspects Med. 18 (Suppl), S7–S13.
48. Morre
´
, D.J., Sedlak, D., Tang, X., Chueh, P.J., Geng, T. & Morre
´
,
D.M. (2001) Surface NADH oxidase of HeLa cells lacks intrinsic
membrane binding motifs. Arch. Biochem. Biophys. 392, 251–256.
49. Morre
´
, D.M., Lenaz, G. & Morre
´
, D.J. (2000) Surface oxidase and
oxidative stress propagation in aging. J. Exp. Biol. 203, 1513–1521.
50. Berridge, M.V. & Tan, A.S. (2000) High-capacity redox control at
the plasma membrane of mammalian cells: trans-membrane, cell
surface, and serum NADH-oxidases. Antioxid. Redox Signal. 2,
231–242.
51. McArdle, A., Pattwell, D., Vasilaki, A., Griffiths, R.D. & Jackson,
M.J. (2001) Contractile activity-induced oxidative stress: cellular

origin and adaptive responses. Am. J. Physiol. Cell Physiol. 280,
C621–C627.
52. Bassenge, E., Sommer, O., Schwemmer, M. & Bu
¨
nger, R. (2000)
Antioxidant pyruvate inhibits cardiac formation of reactive oxy-
gen species through changes in redox state. Am.J.Physiol.Heart
Circ. Physiol. 279, H2431–H2438.
53. Medina, M.A., del Castillo-Olivares, A. & Nunez de Castro, I.
(1997) Multifunctional plasma membrane redox systems. Bioes-
says 19, 977–984.
54. Desai, V.G., Weindruch, R., Hart, R.W. & Feuers, R.J. (1996)
Influences of age and dietary restriction on gastrocnemius electron
transport system activities in mice. Arch. Biochem. Biophys. 333,
145–151.
55. de Grey, A.D.N.J. (2001) A proposed mechanism for the lowering
of mitochondrial electron leak by caloric restriction. Mitochon-
drion 1, 129–139.
56. Herrero, A. & Barja, G. (1997) ADP-regulation of mitochondrial
free radical production is different with complex I- or complex
II-linked substrates: implications for the exercise paradox and
brain hypermetabolism. J. Bioenerg. Biomembr. 29, 241–249.
57. Marklund, S.L. (1984) Extracellular superoxide dismutase and
other superoxide dismutase isoenzymes in tissues from nine
mammalian species. Biochem. J. 222, 649–655.
58. Morre
´
, D.J., Pogue, R. & Morre
´
, D.M. (1999) A multifunctional

hydroquinone oxidase of the external cell surface and sera. Bio-
factors 9, 179–187.
59. Lopez, M.E., Van Zeeland, N.L., Dahl, D.B., Weindruch, R. &
Aiken, J.M. (2000) Cellular phenotypes of age-associated skeletal
muscle mitochondrial abnormalities in rhesus monkeys. Mutat.
Res. 452, 123–138.
60. Appleby, R.D., Porteous, W.K., Hughes, G., James, A.M.,
Shannon, D., Wei, Y.H. & Murphy, M.P. (1999) Quantitation
and origin of the mitochondrial membrane potential in
human cells lacking mitochondrial DNA. Eur. J. Biochem. 262,
108–116.
61. Nagley, P., Farrell, L.B., Gearing, D.P., Nero, D., Meltzer, S. &
Devenish, R.J. (1988) Assembly of functional proton-translocat-
ing ATPase complex in yeast mitochondria with cytoplasmically
synthesized subunit 8, a polypeptide normally encoded within the
organelle. Proc. Natl Acad. Sci. USA 85, 2091–2095.
62. de Grey, A.D.N.J. (2000) Mitochondrial gene therapy: an arena
for the biomedical use of inteins. Trends Biotechnol. 18, 394–399.
63. de Grey, A.D.N.J. (2001) Response to ÔApproaches and limita-
tions to gene therapy for mitochondrial diseasesÕ. Antioxid. Redox
Signal. 3, 1153–1154.
2008 A. D. N. J. de Grey (Eur. J. Biochem. 269) Ó FEBS 2002
64. Zullo, S., Eisenstadt, J.M., Parks, W.T., Fenton, W. & Merril,
C.R. (2000) Gene therapy of the mitochondrial genome: a tale of
two genomes. 2nd NIH Mitochondria Minisymposium, pp. 12–
13. National Institutes of Health, />mito/webversion.pdf
65. Manfredi, G., Fu, J., Sadlock, J.E., Kwong, J.Q., Guy, J. & Schon,
E.A. (2001) Allotopic expression of human ATPase6 in NARP
mutated cells. Mitochondrion 1 (Suppl. 1), S24.
66. Funes, S., Davidson, E., Claros, M.G., van Lis, R., Pe

´
rez-
Martı
´
nez, X., Va
´
squez-Acevedo, M., King, M.P. & Gonza
´
lez
Halphen, D. (2002) The typically mtDNA-encoded ATP6 subunit
of the mitochondrial F
1
F
0
-ATPase is encoded by a nuclear gene in
Chlamydomonas reinhardtii. J. Biol. Chem. 277, 6051–6058.
67. Kolesnikova, O.A., Entelis, N.S., Mireau, H., Fox, T.D., Martin,
R.P. & Tarassov, I.A. (2000) Suppression of mutations in
mitochondrial DNA by tRNAs imported from the cytoplasm.
Science 289, 1931–1933.
68. Seo, B.B., Wang, J., Flotte, T.R., Yagi, T. & Matsuno-
Yagi, A. (2000) Use of the NADH-quinone oxidoreductase
(NDI1) gene of Saccharomyces cerevisiae as a possible cure
for complex I defects in human cells. J. Biol. Chem. 275, 37774–
37778.
Ó FEBS 2002 The reductive hotspot hypothesis (Eur. J. Biochem. 269) 2009