R.M. Anson - Academia.edu (original) (raw)

Papers by R.M. Anson

Mutation Research/DNAging, 1995

The primary focus of this rcvicw ix on correlations found bctwcen DNA damage, repair. and aging. ... more The primary focus of this rcvicw ix on correlations found bctwcen DNA damage, repair. and aging. New techniques for the mcasurcmcnt of DNA damage and repair at the level of individual genes. in individual DNA strands and in individual nucleotides will allow us to gain information regarding the nature of these correlations. Fine structure studies of DNA damage and repair in specific regions, including active gents, telomeres, and mitochondria have begun. Considcrablc intragcnomic DNA repair hctcrogcncity has been found, and there have been indications of relationships between aging and repair in specific regions. More studies are necessary, however. particularly studies of the repair of cndogenous damage. It is emphasized that the information obtained must be viewed from a perspective that takes into account the total rcsponscs of the cell to damaging cvcnts and the interrelationships that exist bctuecn DNA repair and transcription.

DNA Repair Protocols

The Southern blot gene-specific DNA damage and repair assay is a robust and flexible method for q... more The Southern blot gene-specific DNA damage and repair assay is a robust and flexible method for quantifying many kinds of induced damage and repair with high reproducibility. Specific nicking and loss of a restricted DNA fragment at the site of induced damage is visualized by Southern blot and quantified against a control; since the blot is gene specific, only the damage of interest is measured. Here we show how the assay may be adapted to assess mitochondrial DNA (mtDNA) damage. In the mitochondrion, 8-oxoguanine is a significant oxidative lesion; in the laboratory, photoactivated methylene blue may be used to introduce this lesion into cells. Other lesions may also be studied by using different DNA damaging agents. We find that damage induction by methylene blue is consistently far greater in the mitochondrion than the nucleus. Thus advantageously, mitochondrial 8-oxoguanine repair may be studied without mtDNA isolation or preparation, which are processes known to induce DNA damage and skew measurements. This chapter gives detailed instructions for using methylene blue and the gene-specific repair assay to accurately measure mitochondrial oxidative damage and repair rates.

Mutation Research/DNA Repair, 1996

This study examines the capacity of a mammalian cell to repair, at the gene level, DNA base lesio... more This study examines the capacity of a mammalian cell to repair, at the gene level, DNA base lesions generated by photoactivation of acridine orange. Chinese hamster ovary fibroblasts were exposed to acridine orange and visible light, and gene-specific DNA repair was measured in the dihydrofolate reductase (DHFR) gene and in the mitochondrial genome. DNA lesions were recognized by Escherichia co/i formamidopyrimidine-DNA glycosylase (FPG) which removes predominantly 8-oxodG and the corresponding formamidopyrimidine ring opened bases, and subsequently cleaves the DNA at the resulting apurinic site. FPG-recognized DNA lesions increased linearly with increasing photo-activation of AO, while cell survival was not affected by light alone and was negligibly affected by preincubation with A0 in the dark. The frequency of induction of FPG-sensitive DNA damage by photoactivation of A0 was similar in the transcribed and non-transcribed nuclear DNA as well as in the mitochondrial DNA. FPG-sensitive sites in the DHFR gene were repaired quickly, with 84% of adducts repaired within 4 h. The lesion frequency, kinetics and percent of repair of non-transcribed genomic DNA did not differ significantly from repair in the active DHFR gene up to 1 h postexposure. At late time points, transcribed DNA was repaired faster than the non-transcribed DNA. Mitochondrial DNA was efficiently repaired, at a rate similar to that in the active nuclear DNA.

Toxicology Letters, 1998

Living organisms are constantly exposed to oxidative stress from environmental agents and from en... more Living organisms are constantly exposed to oxidative stress from environmental agents and from endogenous metabolic processes. The resulting oxidative modifications occur in proteins, lipids and DNA. Since proteins and lipids are readily degraded and resynthesized, the most significant consequence of the oxidative stress is thought to be the DNA modifications, which can become permanent via the formation of mutations and other types of genomic instability. Many different DNA base changes have been seen following some form of oxidative stress, and these lesions are widely considered as instigators for the development of cancer and are also implicated in the process of aging. Several studies have documented that oxidative DNA lesions accumulate with aging, and it appears that the major site of this accumulation is mitochondrial DNA rather than nuclear DNA. The DNA repair mechanisms involved in the removal of oxidative DNA lesions are much more complex than previously considered. They involve base excision repair (BER) pathways and nucleotide excision repair (NER) pathways, and there is currently a great deal of interest in clarification of the pathways and their interactions. We have used a number of different approaches to explore the mechanism of the repair processes, and we are able to examine the repair of different types of lesions and to measure different steps of the repair processes. Furthermore, we can measure the DNA damage processing in the nuclear DNA and separately, in the mitochondrial DNA. Contrary to widely held notions, mitochondria have efficient DNA repair of oxidative DNA damage and we are exploring the mechanisms. In a human disorder, Cockayne syndrome (CS), characterized by premature aging, there appear to be deficiencies in the repair of oxidative DNA damage in the nuclear DNA, and this may be the major underlying cause of the disease.

Journal of Anti-Aging Medicine, 1999

Free Radical Biology and Medicine, 1999

Mitochondrial and nuclear DNA were isolated from the livers of young (6-7 month) and old (23-24 m... more Mitochondrial and nuclear DNA were isolated from the livers of young (6-7 month) and old (23-24 month) Wistar rats and the levels of 10 different oxidatively induced lesions were analyzed by gas chromatography/mass spectrometry. This is the first study to measure several different oxidatively induced base lesions in both mitochondrial and nuclear DNA as a function of age. No significant age effects were observed for any lesion. Furthermore, contrary to expectations, we did not observe elevated levels of oxidatively induced base lesions in mitochondrial DNA. This contrasts with 50-fold differences reported for several lesions between mitochondrial and nuclear DNA from porcine liver (Zastawny et al., Free Radic. Biol. Med. 24:722-725, 1998). The fact that different lesion levels are observed even when similar techniques are employed emphasizes that the role of oxidative mitochondrial DNA damage and its repair in aging must continue to be the subject of intense investigation. Questions concerning endogenous levels of damage should be revisited as existing methods are improved and new methods become available.

Experimental Gerontology, 2004

Brain Research, 1992

In the striatum and hippocampus, there is a loss of sensitivity to muscarinic agonists with age w... more In the striatum and hippocampus, there is a loss of sensitivity to muscarinic agonists with age which has been traced to events early in the signal transduction pathway. Our laboratory has therefore focussed on investigations at this level. The current experiments investigate the effects of age on G-protein/receptor interactions by using competitive binding assays to measure the ability of GppNHp to decrease the proportion of receptors bound to G-proteins in the absence and the presence of added Mg2+. L-[3H]Quinuclidinyl benzilate was used as a nonselective ligand and [3H]pirenzepine as an M1 selective ligand. We find that: (1) muscarinic receptors and G-proteins in the striatum appear to become loosely coupled with age, with no change in Mg2+ sensitivity. (2) M1-receptor/G-protein complexes in the hippocampus display increased sensitivity to the presence of Mg2+ with age, with those from old but not young tissue requiring added Mg2+ in order to uncouple. This effect, however, may not be M1 specific.

Free Radical Research, 1998

There is an age-associated decline in the mitochondrial function of the Wistar rat heart. Previou... more There is an age-associated decline in the mitochondrial function of the Wistar rat heart. Previous reports from this lab have shown a decrease in mitochondrial cytochrome c oxidase (COX) activity associated with a reduction in COX gene and protein expression and a similar decrease in the rate of mitochondrial protein synthesis. Damage to mitochondrial DNA may contribute to this decline. Using the HPLC-Coularray system (ESA, USA), we measured levels of nuclear and mitochondrial 8-oxo-2&amp;amp;amp;amp;amp;#39;-deoxyguanosine (8-oxodG) from 6-month (young) and 23-month-old (senescent) rat liver DNA. We measured the sensitivity of the technique by damaging calf thymus DNA with photoactivated methylene blue for 30s up to 2h. The levels of damage were linear over the entire time course including the shorter times which showed levels comparable to those expected in liver. For the liver data, 8-oxodG was reported as a fraction of 2-deoxyguanosine (2-dG). There was no change in the levels of 8-oxodG levels in the nuclear DNA from 6 to 23-months of age. However, the levels of 8-oxodG increased 2.5-fold in the mitochondrial DNA with age. At 6 months, the level of 8-oxodG in mtDNA was 5-fold higher than nuclear and increased to approximately 12-fold higher by 23 months of age. These findings agree with other reports showing an age-associated increase in levels of mtDNA damage; however, the degree to which it increases is smaller. Such damage to the mitochondrial DNA may contribute to the age-associated decline in mitochondrial function.

Nucleic acids …, 1998

Photoactivated methylene blue was used to damage purified DNA and the mitochondrial DNA (mtDNA) o... more Photoactivated methylene blue was used to damage purified DNA and the mitochondrial DNA (mtDNA) of human fibroblasts in culture. The primary product of this reaction is the DNA lesion 7-hydro-8-oxodeoxyguanosine (8-oxo-dG). The DNA damage was quantitated using Escherichia coli formamidopyrimidine DNA glycosylase (Fpg) in a gene-specific damage and repair assay. Assay conditions were refined to give incision at all enzyme-sensitive sites with minimal non-specific cutting. Cultured fibroblasts were exposed to photoactivated methylene blue under conditions that would produce an average of three oxidative lesions per double-stranded mitochondrial genome. Within 9 h, 47% of this damage had been removed by the cells. This removal was due to repair rather than to replication, cell loss or degradation of damaged genomes. The rate of repair was measured in both DNA strands of the frequently transcribed ribosomal region of the mitochondrial genome and in both strands of the non-ribosomal region. Fpg-sensitive alkali-resistant oxidative base damage was efficiently removed from human mtDNA with no differences in the rate of repair between strands or between two different regions of the genome that differ substantially with regard to transcriptional activity.

Aging Cell, 2004

Virtually every model of mitochondrial involvement in aging shares the underlying proposition tha... more Virtually every model of mitochondrial involvement in aging shares the underlying proposition that mitochondrial dysfunction will accelerate the rate of aging. Caenorhabditis elegans is a post-mitotic organism with limited capacity for replacement and repair, and there is a great deal of evidence that interventions which decrease the induction of damage extend lifespan in this model. However, decreased availability of ubiquinone in adulthood has also been found to promote longevity and stress resistance, and evidence tentatively supports decreased mitochondrial function under these conditions. In addition, gene silencing experiments and mutations that target mitochondrial electron transport have also been found to increase lifespan and stress resistance in C. elegans , as has treatment with the mitochondrial inhibitor antimycin A. The involvement of damage by reactive oxygen species has been suggested, and yet many of these manipulations would be expected to increase the production of reactive oxygen species. The extension of lifespan by these interventions seems paradoxical and the mechanism, when it is elucidated, promises to have far-reaching significance.