Oxidative damage to mitochondrial DNA and its relationship to ageing

hr. J. Biochem.

Pergamon

1357-2725(95)00025-9

Cell Bid. Vol. 27, No. 7, pp. 641653.

1995

Copyright 0 1995 Elsevier Science Ltd Printed in Great Britain. All rights reserved 1357-2’725/95

1129.00 i- 0.00

MINIREVIEW

Oxidative Damage to Mitochondrial ~e~~ti~~~~ to Ageing CHRISTOPH Laboratory CH-8092

DNA and its

RICHTER

of Biochemistry I, Swiss Federal Institute Ziirich, Switzerland

of Technology (ETH), Universitiitstr.

16,

Mitochodria are the most important intracellular source of reactive oxygen speciespd are protected @ast them by enzymatic ad nowazymptjc m%ti~xidmttS.Nevea DNA (mtDNA) is sabject to severe oxidntive dunage, and much more so l&811 (nDNA). Damage is indicated by the detection of various base modification, 8-hydroxydeoxygumoshe @OHdG), which can lead to pohtt ldep MtDNA is also fragmeuted to some extent. C deletions found itl mtDNA. Severalhypothesessu or are responsiblefor, agehq& Recent observationsindiWe that mito&mkia in an old differ in many respec@from those in a yomkgorganism. Thm with piecingthere is an hWeased prodtsction of reactive oxygen saecies, a decrease in certain antioxidancS, a decrensed traeNA scription, t3aashstiou, ad cytochrome oxidase codeot, and an increase in the ex we mod&cations. Major unresolved questioas cwceraiag the role of t&DNA char&es m atidmsd is there a causal relationship; what is the true extent of DNA dnmage; what are the oxygen speciesthe SigniEcanceaud fwn&onal consequencesof mtDNA between DNA damage cause of the DNA modifications found in uiuo; wbat awl alterations of RNAs and protehs? Future studies promise to clrriry the poasibk cmtsal relationship between mitochondrial dysfonction, reactive oxygen species p&u&on, mtDNA moditkations, and age@. Keywords: Oxygen radicals Mitochondrial DNA

Deletions Mutations

Ageing

Int. J. Biochem. Cell Biol. (1995) 27, 647-653

post-translational modifications, quantitative changes of proteins, translational changes, According to Medvedev’s estimate (1990) changes in.DNA and RNA, and changes at the there are more than 300 theories of ageing, organ or functional level. At present the most many of which co-exist because they do not popular and widely tested damage theory is the contradict each other, or because they try free radical theory of ageing, first proposed by to explain different and independent forms Harman (1956). More recently this theory of senescence. An important group of ageing focused on oxygen radical production in mitotheories originates from the study of changes chondria. As outlined below, evidence accumuthroughout life or changes which accumulate in lated during the last six years which suggests time. These theories encompass cross-linkage, that oxidative modifications of mtDNA may be of central importance in normal and pathoAbbreviations: mtDNA, mitochondrial DNA; nDNA, logical ageing. nuclear DNA; 80HdG, 8-hydroxygeoxygnanosine; This article first describes the prooxidative PCR, polymerasechain reaction; ROS,reactiveoxygen and antioxidative capacities of mitochondria, species. and properties of mtDNA. Most of the original Received23 June 1994;accepted1 March 1995. INTRODUCTION

K 2717-B

647

648

Christoph Richter

references to these topics can be found in recent reviews (Bandy and Davison, 1990; Richter, 1992).* Ageing theories based on mitochondrial alterations are then briefly outlined. Next, the most recent data on modifications emphasising oxidative damage to mtDNA, and their relationship to ageing are reviewed. Finally, major unresolved questions are posed and future studies suggested.

MITOCHONDRIAL

DNA

Mitochondria originate from symbiotic bacteria. These organelles contain therefore their own DNA, with many peculiar properties. During evolution, genes were transferred from mitochondria to the nucleus, and the size and coding capacities of mtDNA were thereby reduced. The remaining 16,596 base-pairs of human mtDNA code for two ribosomal RNAs, 22 transfer RNAs, and 13 peptides which are REACTIVE OXYGEN SPECIES IN part of five multi-subunit enzymes of the oxiMITOCHONDRIA dative phosphorylation machinery in the inner Reactive oxygen species (ROS) such as super- mitochondrial membrane. The universal genetic oxide radical, hydrogen peroxide, hydroxyl code is used without changes in mitochondria of radical, and singlet oxygen are products of green plants, but non-plant mitochondria use normal metabolism. Some compounds (e.g. a code which is slightly different. so-called “redox cyclers” such as paraquat, Cells can contain up to about one thousand adriamycin, mitomycin C, 6-hydroxydopamine, mitochondria, each carrying five to ten mtDNA alloxan, nitrofurantoin, metronidazole or bleo- molecules. In mammals, mtDNA mutates mycin) increase their production, as do some much faster than nDNA. Expression of variant pathological states (e.g. inflammation, iron mtDNA depends on the extent of segregation overload, adult respiratory dystress syndrome of heteroplasmic (mixture of normal and muor intoxication with “redox cyclers”). Mitotant) mtDNAs and the need for mitochondrial chondria consume about 90% of the oxygen function. Mitochondria are almost exclusively used by the body, and are a particularly maternally inherited. They proliferate indepenrich source of ROS since about l-2% of dently of the cell cycle. In adult rats mtDNA oxygen consumed by mitochondria is converted has a half-life between several days and a to superoxide and hydrogen peroxide. For month, depending on the organ (Gross et al., example, one rat liver mitochondrion produces 1969). about 3 x 10’ superoxide radicals per day. Mitochondrial DNA is not covered by Several sites of the mitochondrial respiratory histones, and is at least transiently attached chain and matrix are constitutive generators to the inner mitochondrial membrane, which of ROS. Alloxan, menadione, rotenone, produces large amounts of ROS. Therefore, methylphenylpyridinium, tetrachloro-dibenzomtDNA is particularly susceptible to oxidative p-dioxin, elevated Ca2+, or tumour necrosis damage. For example, the steady-state level of factor-a stimulate ROS production by mitooxidised bases in the mtDNA of 3 month-old chondria, as does hypoxia/reperfusion. rats is about 16 times higher than in nDNA In mitochondria superoxide and hydrogen (see also below). peroxide are metabolised by the Mn-containing Because mtDNA has very little redundancy superoxide dismutase and the Se-containing and a high information density, human diseases glutathione peroxidase, respectively. Furthercaused by mutations in mtDNA could be more, ROS are scavenged by vitamins C and expected, and were indeed identified (Wallace, and ubiquinol-10. Despite E, glutathione, 1992a, b). these efficient antioxidative defence systems, DNA repair in the nucleus is essential for oxidative damage is detectable in mitochondrial survival and evolution (Koshland, 1994). In lipids, proteins, and nucleic acids (Richter ef al., mitochondria, DNA repair is much less efficient 1995). than in the nucleus. The mammalian organelles do not have significant recombinational repair but may excise damaged bases, since they con*After this article had been submitted, a review on the tain uracil DNA glycosylases, AP endonuclerelationship between mitochondria, oxygen, and ageing ases, and U.V. endonucleases. Mitochondria has been published [Shigenaga M. K., Hagen T. M. and repair alkylated DNA bases. The presence of Ames B. N. (1994) Oxidative damage and mitochondrial suggests at decay in aging. Proc. Nam. Acad. Sci. U.S.A. 91, these enzymes in mitochondria least some DNA repair. However, the enzymes 10771-107781.

Oxidative damage to mitochondrial

may also, or additionally, degrade damaged mtDNA in order to assure the survival of only the population of undamaged mitochondrial genomes. Very recently for the first time, evidence for repair of oxidative damage in mtDNA of cultured cells has been presented (Driggers et al., 1993). The technique used for the detection of repair does not require the sometimes tedious isolation of mtDNA and should prove useful in future studies with other cell systems. EXTENSION

OF

MAXIMAL

LIFE

SPAN

A better understanding at the molecular level of ageing can be expected from studies in which the maximal life span is manipulated. With the choice of shortening or extending it, the latter is of course much more revealing. In mammals, there are two established methods to extend maximal life span: restriction of calorie intake and treatment with deprenyl. The former is accompanied by a decreased oxidative damage of both nuclear and mtDNA (see below). The latter may also be related to decreased oxidative stress since deprenyl inhibits monoamine oxidase (type B). This enzyme is localised in the outer mitochondrial membrane and catabolises dopamine, which is present in high millimolar concentrations within the neurone, yielding hydrogen peroxide stoichiometrically. In frogs, induction of various tissue antioxidant enzymes and nonenzymatic antioxidants increases the mean life span (LopezTorres et al., 1993). In flies, reduced physical activity is paralleled by an extension of average and maximal life span, and by decreased protein oxidation (Sohal et al., 1993). AGEING MITOCHONDRIAL

THEORIES DNA

BASED ON ALTERATIONS

Harman (1956) proposed that free radicals play a role in ageing through crosslinking reactions, which could be due to the generation of lipid, protein and DNA radicals followed by the formation of covalent bonds, or due to the reaction of oxidatively formed aldehydes with amino groups. Harman suggested that one of the possible sites of free radical attack is mtDNA of all cell types (Harman, 1983). A similar proposal was made by Miquel and colleagues (Miquel et al., 1980; Miquel, 1991) who proposed that ageing results from changes

DNA

649

of the mitochondrial genome of differentiated cells. Based on the finding that mtDNA is fragmented by ROS, Richter (1988) suggested that mtDNA fragments escape from mitochondria and accumulate in a time-dependent manner in nDNA, which would progressively change the nuclear information content and thereby cause ageing. This hypothesis is consistent with the finding that gene transfer from mitochondria to the nucleus is rapid and essentially a “one-way traffic” in yeast (Thorsness and Fox, 1990). In higher eukaryotes, a thorough analysis is yet to be done. Subsequently, Linnane et al. (1989) suggested that bioenergetically defective cells are a key factor in the ageing process, and that the generation of free radicals in mitochondria could continuously damage mtDNA. According to Kadenbach and Miiller-Hacker (1990) the accumulation during life of respiratory-deficient cells mainly in human heart limits the life-span of each individual. CHANGES

IN

MITOCHONDRIA AGEING

DURING

Respiratory enzyme activities have been investigated in mitochondria isolated from various tissues of species as different as flies, rats, and humans. Most reports indicate a decrease in complex I and, to a lesser extent of complex IV (cytochrome oxidase), activities with age, while complex II and III activities are mostly unaffected. This seems important because many of the subunits of complex I and IV are encoded by mtDNA, whereas complex II is exclusively encoded by nDNA. It should also be noted that there is a decrease of 12s rRNA and the mRNA for subunit II of cytochrome oxidase in brain and heart but not in liver of senescent rats. Also the amount of mitochondrial enzymes changes with age. Histochemical analyses in human heart, diaphragm and limb muscle revealed an age-associated increase of the number of cells lacking cytochrome oxidase. Mitochondria of aged animals produce more ROS than those of young animals. This has been shown for both insects and mammals, species in which maximal life span potential relates inversely to the rate of oxygen consumption and positively to antioxidant capacity. Mitochondria from older rats also show higher levels of lipid peroxides and losses of polyunsaturated fatty acids.

Christoph Richter

650 CHANGES OF MITOCHONDRIAL INCLUDING OXIDATIVE DAMAGE, AGEING

DNA, DURING

Since the first descriptions of mtDNA alterations related to a disease (Holt et al., 1988; Wallace et al., 1988) numerous reports documented the occurrence of point mutations and deletions in various diseases such as blindness, deafness, dementia, movement disorders, weakness, cardiac failure, diabetes, renal dysfunction, liver, Parkinson’s, and Alzheimer’s diseases (for original references the reader is referred to two comprehensive reviews by Wallace, 1992a, b). Mitochondrial gene alterations could also make significant contributions to ageing. Results obtained with various experimental approaches indicate a positive correlation between normal or pathological ageing and mtDNA modifications. The literature up to Spring 1992 has been reviewed (Richter, 1992). Below, only the most recent data on modifications including oxidative damage to mtDNA and their relationship to ageing are considered. Earlier reports indicated that ageing causes a remarkable reduction of mitochondrial gene expression in mammals. More recent results showed that senescent brain cells of rats have a decreased content of the D-loop portion of mtDNA (Petruzzella et al., 1992). The D-loop is an important element in mtDNA replication, and damage to the D-loop can reasonably be expected to be a severe threat to mitochondrial proliferation. In Drosophila, however, Southern blot analysis indicated a high stability and integrity of mtDNA during ageing, whereas the levels of mitochondrial RNAs were reduced (Calleja et al., 1993). Lipid peroxidation in mitochondria, which results in the formation of 80HdG in mtDNA, also causes cross-linking of mtDNA to proteins (Hruszkewycz, 1992). That such a covalent mtDNA modification by proteins may occur during ageing is indicated by the report of Asano et al. (1991) that the buoyant density profile of mtDNA of old rats is broad, but becomes similar to that of mtDNA of young rats after treatment with proteinase K. Furthermore, mtDNA gained resistance against EcoRI digestion during ageing, a property possibly due to the binding of protein(s). If protein binding affects gene expression in mitochondria, it requires further investigation. An age-dependent accumulation of point mutations in human mtDNA has recently been

detected in two independent studies (Miinscher et al., 1993; Zhang et al., 1993). The most widely measured mtDNA modification is 80HdG, since it can be reliably detected in the femtomole range. Its presence in mtDNA was first reported by Richter et al. (1988) who found that in livers of 3 month-old rats, 80HdG is about 16 times more abundant in mtDNA than in nDNA. These findings were recently confirmed and extended by an investigation of DNA damage in old rats (Ames et al., 1993): in addition to the high 80HdG content of mtDNA in the liver of young animals, they found that in 24 month-old animals, oxidative mtDNA damage is 3 times higher, whereas damage of nDNA is doubled in the old animals. These data are partly in conflict with the report of Chung et al. (1992) who did not find a difference in the 80HdG content of 3 and 24 month-old rats. The latter authors also found a 1Cfold higher level of oxidative damage in mtDNA over nDNA. Importantly, they also reported that calorically restricted animals have a significantly lower 80HdG content in both nuclear and mtDNA compared to ad libitum-fed animals. As noted above, calorie restriction results in an increased life expectancy. Hayakawa and co-workers (1991, 1992) showed an age-associated accumulation of this modified base in mtDNA of human diaphragmatic and heart muscle. In the latter tissue mtDNA deletions were highly correlated with the 80HdG content of mtDNA (Hayakawa er al., 1992). Finally, oxidative damage as measured by the 80HdG content increases particularly at old age in human brain (Mecocci et al., 1993). Three regions of cerebral cortex and cerebellum from 10 normal humans aged 42 to 97 yr were investigated. 80HdG levels increased progressively with age in both nDNA and mtDNA, with the rate of increase being much greater in mtDNA. A IO-fold increase in the amount of 80HdG was found in mtDNA as compared to nDNA in the entire group of samples, and a 15-fold increase in subjects over 70 yr. The same group (CorralDebrinski et al., 1992) found an increase of mtDNA deletions in human brain with advancing age. In contrast, Ames and co-workers (Fraga et al., 1990) reported that 80HdG increases in nDNA with ageing in liver, kidney, and intestine, but not in brain or testes of rats. Very recently, the first clear experimental evidence for a causal relationship between

Oxidative damage to mitochondrial

ROS production in mitochondria and mtDNA modifications in vivo was presented. Ada&i et al. (1993) reported that doxorubicin treatment of mice leads to a dose- and timedependent formation of deletions in cardiac mtDNA. Previous studies had shown that doxorubicin stimulates ROS production in mitochondria. Indeed, when Asano et al. used doxorubicin in combination with ubiquinol- 10, a known outstanding antioxidant, the extent was drastically of mtDNA modification reduced. If confirmed, these results would be of paramount importance in understanding the etiology of ageing and age-related degenerative diseases. 80HdG causes mispairing at neighbouring bases and point mutations. This has recently been documented also for the replicative polymerase of mtDNA despite the presence of a highly active proof-reading exonuclease activity (Pavlov et al., 1994). 8OHdG is just one out of many lesions produced by ROS in DNA. Presently it is difficult to assign a particular protein modification to a given mtDNA mutation, but it was shown recently that tandem double CC to TT mutations are produced by ROS (Reid and Loeb, 1993). CONCLUSION

AND FUTURE STUDIES

From the studies discussed above several facts become apparent: l

l

l

l

l

mtDNA is much more susceptible to oxidative damage than nDNA; ROS cause base modifications and strand breaks in mtDNA in vitro, and most likely the occurrence of deletions in vivo; ROS production by mitochondria increases with age; mtDNA modifications increase with age, particularly the levels of SOHdG, a known mutagen; mitochondria in many or all organs of an old individual perform more poorly than those of a young individual.

Major unresolved issues remain. They are addressed in the following sections. Most urgent is to provide conclusive experimental evidence for a causal relationship between mtDNA modifications and ageing, and between mitochondrial ROS production, dysfunction, and ageing. If this can be achieved there will be hope to be able to fight age-related diseases and even extend maximal life span.

DNA

651

WHAT IS THE REAL EXTENT OF MITOCHONDRIAL DNA DAMAGE?

Most investigations of age-dependent mtDNA modifications have concentrated on the levels of 80HdG and the frequency of certain deletions. Both approaches presently do not give quantitative information about the true level of modifications. This is because 80HdG is only one out of 20 to 30 oxidative base modifications identified in model reactions. They indicate that 80HdG is one of the most frequently encountered modified bases, but still it is not clear if these model reactions mimic the situation prevailing inside mitochondria. Other oxidatively modified bases in mtDNA have not been identified to date. Deletions were so far mostly detected by the polymerase chain reaction (PCR). The results are therefore biased by the choice of primers. The same is of course true for PCR studies which search for point mutations, Whereas at least some deletions and point mutations have been quantified we have no knowledge at all about the extent of mtDNA strand breaks and fragmentation. With PCR it is in principle possible to systematically scan the mitochondrial genome for the frequency, size, and position of deletions, and the frequency and position of point mutations. In this way putative hot-spots for modifications could be identified. Further studies could then be directed towards an understanding of the mechanism of their formation. So far, slip mispairing has been suggested to be the basis, but it may well be that certain sites of the mtDNA, possibly due to topological constraints or its attachment at ROS-generating sites in the inner mitochondrial membrane, are particularly sensitive to oxidative attack and therefore prone to mutation. WHAT ARE THE SIGNIFICANCE AND FUNCTIONAL CONSEQUENCES OF MITOCHONDRIAL DNA DAMAGE?

Given the high level of oxidative mtDNA damage it is amazing and unresolved how mitochondria cope with it. This relates to the question of how and to what an extent damage is repaired (maybe it is more economical to make new mitochondria; maybe DNA fragments serve a useful purpose) during the lifetime of a mitochondrion and also to the question how a reasonably intact genome is preserved over the years in an individual, and from generation to generation. Do mitochondrial DNA (and RNA)

652

Christoph Richter

polymerases substrates? ARE

recognise and neglect damaged

ROS THE CAUSE OF DNA MODIFICATIONS

MITOCHONDRIAL

IN VIVO?

Hydroxyl radicals are most likely the agents responsible for oxidative base modifications and strand scission in DNA. They are formed when hydrogen peroxide is reduced, e.g. by superoxide in the presence of heavy metal ions. The study by Hayakawa et al. (1992) shows a close correlation between 80HdG levels and deletions in human mitochondria. Adachi et al. (1993) give the first clear evidence that ROS are responsible for the formation of deletions. Additional studies are needed to establish the relationship between ROS and mtDNA modifications found in uivo. The most promising experimental approach would be to create animals overexpressing mitochondrial superoxide dismutase and glutathione peroxidase. When overexpressed together they should afford protection against damage (and therefore provide longevity?) since their joint action should decrease the steady-state level of hydroxyl radicals. It would also be very interesting to overexpress mitochondrial heme oxygenase and ferritin, proteins which liberate and bind iron ions, respectively. Alternatively it will be of interest to study mtDNA of animals whose Mn-, Se-, and antioxidant vitamin-states are lowered. WHY IS MIT’OCHONDRIAL DETECTABLE ONLY

AT

DNA CERTAIN

DAMAGE AGES?

Although convincing experimental proof is not yet available, ROS can reasonably be assumed to cause the age-related damage to mtDNA. But why is there an age-related and different onset of damage in the various organs (at the age of 35-40 in human heart, at the age of 7@-SOin the brain)? Maybe defence systems active against ROS are exhausted, maybe due to a replicative advantage, deleted mtDNA molecules reach a decisive level, maybe oxidatively induced mutations code for proteins which increase ROS production (see next paragraph). WHAT ARE THE CONSEQUENCES OF DAMAGE FOR RNAS AND PROTEINS MITOCHONDRIA?

DNA IN

The formation of 80HdG is mutagenic. Other base modifications almost certainly

will also alter the information content of mtDNA, as do deletions. Little is known about the consequences at the RNA and protein level. The few results collected so far indicate that transcription is impeded with age, and that those components of the mitochondrial respiratory chain which are coded for by mtDNA are particularly labile with advancing age. It is a future challenge to relate mutations in mtDNA to alterations in mitochondrial proteins, WHICH ORGAN FAILS PHOSPHORYLATION

FIRST IN DURING

OXIDATIVE AGEING?

A decline in oxidative phosphorylation capacity has been proposed to limit life expectancy (Kadenbach and Miiller-Hiicker, 1990; Wallace 1992a, b). Assuming that this is so, it is not clear if the failure of one particular organ causes death. Anatomically normal hearts do not spontaneously fail in old humans, but neurological deficiencies are common in the aged. Future studies should investigate if mtDNA damage is more pronounced in the brain than in the heart, and if there are regionspecific modifications in brain mtDNA which correlate with age-related deficiencies. Acknowledgements-I thank Drs Schweizer, Suter, and Walter for critically reading the manuscript and helpful suggestions.

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