Mutation Research/DNAging最新文献

We have observed and characterized in detail two cases of mitochondrial DNA fragments which have inserted into the nucleus of HeLa cells. In one case three non-sequential but contiguous regions of mitochondrial DNA with 92% homology to human cytoplasmic mitochondrial DNA inserted into the nuclear genome. In the second case the mitochondrial DNA sequence encoding cytochrome c oxidase subunit III was contiguous with and 5′ of exons 2 and 3 of the c-myc oncogene and the chimeruc gene was transcribed. Models are presented that describe mechanisms for the transfer of mitochondrial DNA into the nucleus involving fragmentation of mitochondrial DNA through aging and/or oxidative damage, anomalous processing or escape of mitochondrial DNA and RNA fragments from autophagic vacuoles, and insertion of mitochondrial DNA sequences, in some instances after reverse transcription of mitochondrial RNA, into the nuclear genome.

Mitochondria are the major intracellular producers of O₂⁻ and H₂O₂. The level of oxidative stress in cells, as indicated by the in vivo exhalation of alkanes and the concentration of molecular products of oxy-radical reactions, increases during aging in mammals as well as insects. In this paper, we discuss the relationship between mitochondrial generaton of O₂⁻ and H₂O₂, and the aging process. The rate of mitochondrial O₂⁻ and H₂O₂ generation increases with age in houselifes and the brain, heart and liver of rat. This rate has been found to correspond to the life expectancy of flies to the maximum life span potential (MLSP) of six different mammalian species, namely, mouse, rat, guinea pig, rabbit, pig and cow. In contrast, the level of antioxidant defenses provided by activities of superoxide dismutase, catalase, glutathione peroxidase and glutathione concentration neither uniformly declines with age nor corresponds to variations in MLSP of different mammalian species. It is argued that the rate of mitochondrial O₂⁻ and H₂O₂ generation rather than the antioxidant level may act as a longevity determinant.

The mitochondrial respiratory chain and oxidative phosphorylation system are responsible for the production of ATP by aerobic metabolism. Defects of the respiratory chain are increasingly recognised as important causes of human disease, and neurodegenerative disorders in particular. This article will seek to review the clinical and biochemical effects of respiratory chain defects, and summarise what is known about the molecular mechanisms that underlie them. Increasing age is also associated with a decline in mitochondrial function. The biochemical correlates of this dysfunction and the possible molecular defects that may cause it will also be reviewed.

Efforts have been made to characterize and measure DNA modifications produced in mammalian chromatin in vitro and in vivoby a variety of free radical-producing systems. Methodologies incorporating the technique of gas chromatography/mass spectrometry have been used for this purpose. A number of products from all four DNA bases and several DNA-protein cross-links in isolated chromatin have been identified and quantitated. Product formation has been shown to depend on the free radical-producing system and the presence or absence of oxygen. A similar pattern of DNA modifications has also been observed in chromatin of cultured mammalian cells treated with ionizing radiation or H₂O₂ and in chromatin of organs of animals treated with carcinogenic metal salts.

To study the interaction of singlet oxygen (¹O₂) with DNA and the biological consequences of ¹O₂-induced DNA damage, we used the thermodissociable endoperoxide of 3,3′-(1,4 naphtalidene) dipropionate (NDPO₂) as a generator of free ¹O₂ in reactions with (1) 2′-deoxynucleoside 3′-monophosphates (dNps), (2) an oligonucleotide (16-mer) having one deoxyguanine (dG), (3) native and denaturated rat kidney DNA and (4) single-stranded (ss) and double-stranded (ds) bacteriophage M13mp10 DNA. Using both anion exchange and reversed phase HPLC and ³²P-postlabeling analyses, it was found that exposure of the various dNps to chemically generated ¹O₂ led to a detectable reaction with dGp and not with dAp, dCp, d5mCp or Tp. The reaction with dGp led to degradation of this nucleotide and the formation of a large number of reaction products, one of which could be identified as 7-hydro-8-oxo-2′-deoxyguanosine 3′-monophosphate (8-oxo-dGp).

A second product could tentatively be identified as a formamido pyrimidine derivative of dGp (Fapy-dGp). When ss DNA, ds DNA or the oligonucleotide were exposed to ¹O₂, the formation of 8-oxo-dG could also be demonstrated. With the oligonucleotide, we found a so far unidentifed reaction product. Under the same reaction conditions the yield of 8-oxo-dG was about 8-fold higher in ss DNA than in ds DNA. In ss DNA 8-oxo-dG seemed to be a more prominent product than in the case of reaction of ¹O₂ with free dGp.

Reaction of ¹O₂ with ss or ds M13mp10 DNA led to biological inactivation of these DNAs, ss DNA being at least 100-fold more sensitive than ds DNA. It could be concluded that inactivation of the ss DNA must be largely due to ¹O₂-induced DNA lesions other than 8-oxo-dG. In agreement with the observed preferential reaction of ¹O₂ with dG most of the so far sequenced mutations, induced by ¹O₂ in a 144 bp mutation target sequence inserted in the lacZα gene of ss or ds M13mp10 DNA, occurred at a

We review the role that oxidative damage plays in regulating the lifespan of the fruit fly, Drosophila melanogaster. Results from our laboratory show that the lifespan of Drosophila is inversely correlated to its metabolic rate. The consumption of oxygen by adult insects is related to the rate of damage induced by oxygen radicals, which are purported to be generated as by-products of respiration. Moreover, products of activated oxygen species such as hydrogen peroxide and lipofuscin are higher in animals kept under conditions of increased metabolic rate. In order to understand the in vivo relationship between oxidative damage and the production of the superoxide radical, we generated transgenic strains of Drosophila melanogaster that synthesize excess levels of enzymatically active superoxide dismutase. This was accomplished by P-element transformation of Drosophila melanogaster with the bovine cDNA for CuZn superoxide dismutase, an enzyme that catalyzes the dismutation of the superoxide radical to hydrogen peroxide and water. Adult flies that express the bovine SOD in addition to native Drosophila SOD are more resistant to oxidative stresses and have a slight but significant increase in their mean lifespan. Thus, resistance to oxidative stress and lifespan of Drosophila can be manipulated by molecular genetic intervention. In addition, we have examined the ability of adult flies to respond to various environmental stresses during senescence. Resistance to oxidative stress, such as that induced by heat shock, is drastically reduced in senescent flies. This loss of resistance is correlated with the increase in protein damage generated in old flies by thermal stress and by the insufficient protection from cellular defense systems which includes the heat shock proteins as well as the oxygen radical scavenging enzymes. Collectively, results from our laboratory demonstrate that oxidative damage plays a role in governing the lifespan of Drosophila during normal metabolism and under conditions of environmental stress.

A survey of the main available chemical and biochemical postlabeling assays for measuring oxidative DNA damage is reported. Two main approaches, radio and fluorescent postlabeling, have been used in order to reach a high level of sensitivity of detection. This is required for the measurement of DNA damage within cells and tissues upon exposure to agents of oxidative stress. Most of the methods are based on liquid chromatographic separation of defined DNA modifications following either acidic hydrolysis or enzymic digestion of DNA. In a subsequent step, the isolated base or sugar damages are either radiolabeled or made fluorescent by chemical or enzymatic reactions. Emphasis is placed on the recently developed high performance liquid chromatographic ³²P-postlabeling assay, which allows the specific and sensitive measurement of various base damages including adenine N-1 oxide and 5-hydroxy-methyluracil at the level of one modification per 10⁷ normal bases in a sample size of 1 μg of DNA. Examples of application of radioactive postlabeling to the measurement of DNA base damage following exposure of human cells to oxidizing agents including hydrogen peroxide and UVA radiation are provided.

During the last decade the importance of reactive oxygen species as major contributors to various types of cancer, heart diseases, cataracts, Parkinson's and other degenerative diseases that come with age, and to natural aging has become apparent. Mitochondria are the most important intracellular source of reactive oxygen. Mitochondrial DNA is heavily damaged by reactive oxygen at the bases, as indicated by the high steady-state level of 8-hydroxydeoxyguanosine, the presence of which causes mispairing and point mutations. Mitochondrial DNA is also oxidatively fragmented to a certain extent. Conceivably, such fragmentation relates to deletions found in mitochondrial DNA. Point mutations and deletions have recently been shown to be etiologically linked to several human diseases and natural aging. Future studies should address the causal relationship between mitochondrial dysfunction, production of reactive oxygen species, and aging.

End-products of lipid peroxidation accumulate during the life of somatic cells. It is hypothesized that genotoxic intermediates of lipid peroxidation may have a role in causing age-associated DNA mutations. Such mutations are likely to accrue in the mitochondrial genome because it, unlike nuclear DNA, is not protected by histones and repair systems. In addition, it is located near the mitochondrial membrane where lipid peroxidation can be initiated by free radicals produced by the mitochondrial electron transport system. This idea is supported by in vitro experiments which show that mitochondrial DNA is damaged when mitochondria undergo lipid peroxidation.

The aim of our study was first to obtain a comprehensive profile of the brain antioxidant defense potential and peroxidative damage during aging. We investigated copper-zinc superoxide dismutase (CuZnSOD), manganese superoxide dismutase (MnSOD), seleno-dependent glutathione peroxidase (GSH-PX), glutathione reductase (GSSG-R) activities, endogenous and in vitro stimulated lipid perxidation in 40 brains of control mice divided into 3 age groups: 2 months (young), 12 months (middle-aged) and 28 months (old). We found a positive correlation between age and activities of CuZnSOD (r = 0.47); P < 0.01) and GSH-PX (r = 0.72; P < 0.0001). CuZnSOD and GSH-PX activities are independently regulated during brain aging since temporal changes of these two enzymes do not correlate. No modification in MnSOD activity and basal lipid peroxidation was observed as a function of age. Nevertheless, stimulated lipid peroxidation was significantly higher at 12 months (6.53 ± 0.71 μmole MDA/g tissue) thatn at 2 months (5.69 ± 0.90) and significantly lower than 28 months (5.13 ± 0.33) than at 12 months.

Second, we used genetic manipulations to construct transgenic mice that specifically overexpress CuZnSOD to understand the role of CuZnSOD in neuronal aging. The human CuZnSOD transgene expression was stable during aging. The increased CuZnSOD activity in the brain (1.9-fold) of transgenic mice resulted in an enhanced rate of basal lipid peroxidation and in increased MnSOD activity in the 3 age groups. Other antioxidant enzymes did not exhibit modifications indicating the independence of the regulation between CuZnSOD and glutathione-related enzymes probably due to their different cellular localization in the brain.