Mutation Research/DNAging最新文献

We are constantly exposed, throughout life, to a wide variety of extrinsic and intrinsic agents which have the potential to damage cellular biomoleculcs, including DNA. Imperfections in cellular defence systems which protect against the fixation of DNA damage can lead to an accumulation of mutations which on their own, or in combination with other age-related changes, may contribute to ageing and the development of age-related pathologies. We have previously reported an increase in frequency of mutation with age in human lymphocytes taken from healthy males in the age groups, 35–39. 50–54 and 65–69 years. In this article we report on the findings of a recent study which was designed to assess whether the age-related increase in frequency of mutation was due to a decreased efficacy of the defence systems against ROS-induced DNA damage, namely antioxidant status and DNA repair processes, in the same study subjects. In vivo antioxidant status was assessed in each of the study subjects by measuring blood levels of; superoxide dismutase (SOD; EC 1.15.1.1), glutathionc peroxidase (GPx; EC 1.11.1.9), catalase (EC 1.11.1.6), caeruloplasmin (CPL), uric acid and bilirubin. We did not find any statistically significant differences in the mean levels of these antioxidants between the three different age groups. To investigate the efficacy of DNA repair processes against ROS-induccd DNA damage, an ELISA was used to quantitate DNA damage (as % single-stranded DNA; %SS-DNA) at various times following treatment of peripheral blood lymphocytes with hydrogen peroxide (H₂O₂). The results of this part of the study showed that in untreated lymphocytes, basal levels of %SS-DNA were significantly higher in individuals from the 65–69 years age group compared to the 35–39 years age group (p = O.039, 0.0013; at 5% level of significance). No significant differences were found in H₂O₂ susceptibility with age immediately following treatment (p = 0.71, 1.00; at 5% level of significance) but a consistent and significant increase was observed in %SS-DNA remaining 90 min post-treatment in lymphocytes from subjects in the 65–69 years age group, compared to %SS-DNA present in lymphocytes from the 35–39 years age group (p = 0.013, 0.024; at 5% level of significance).

The results of this study suggest that the age-related increase in frequency of mutations is not contributed to by alterations of in vivo antioxidant status with age but is by a decreased efficacy of the repair of ROS-induced DNA damage with age. The biological implications of somatic mutations in the ageing process are discussed.

Using either the colony formation assay or flow cytometry, it is feasible to measure the frequency of rare mutant lymphocytes or erythrocytcs in human peripheral blood. Accordingly, we have investigated the mutant cell frequencies of the hypoxanthine-guanine phosphoribosyltransferase and T-cell receptor genes in T lymphocytes and of the glycophorin A gene in erythrocytes of several hundred persons aged 0–96 years. The mutant frequency of every one of these genes increased significantly with age. A simple accumulation of mutations in hematopoietic stem cells over time may explain the age-dependent increase in the frequency of glycophorin A mutants. In contrast, a balance between mutant cell generation and loss should be taken into account for the mechanism of the increase of T-cell mutations.

Theories on the causes of aging, based on the accumulation of somatic mutations in tissues of an organism, were formulated decades ago, but remain insufficiently tested. Transgenic animals, equipped with integrated bacterial reporter genes that can be efficiently rescued from total genomic DNA of all tissues and organs, represent ideal tools for investigating the types and frequencies of spontaneous mutants accumulating during aging. The first of such systems, based on the transgenic integration of bacteriophage lambda shuttle vectors that contain the bacterial lacZ gene as mutational target, was constructed in our laboratory and is now routinely used. Results obtained with this and the related LacI system that are relevant for the somatic mutation theory of aging will be discussed. One conclusion is that, due to the nature of the transgene. lambda-based systems have the disadvantage that deletion type mutations are underrepresented in comparison to point mutations. To overcome those limitations, we constructed a new transgenic mouse model carrying a pUR288 plasmid shuttle vector with the lacZ reporter gene. Some preliminary data obtained with this model serve to illustrate its potential use to extensively test the somatic mutation theory of aging.

Analysis of two human populations for dependence of somatic mutation on age has revealed both similarities and differences. The studies performed employed peripheral blood lymphocytes and measured the efficiency with which these cells form clones in vitro (cloning efficiency, CE) and the frequency of cells with inactivating mutations of the hypoxanthine phosphoribosyltransferase gene (mutant frequency, MF). The people studied were between 19 and 64 years of age. In one population, composed of 78 never smokers and 140 current smokers from the United States (US), both CE and MF were dependent on age: CE declined with age (p = 0.005); MF increased 0.15 per 10⁶ cells per year of age for nonsmokers (p < 0.001) and at 1.3 times that rate for smokers (p = 0.01). In the second population, 80 people of unknown smoking status from Russia, the increase in MF per year was even greater, 2.5 times that of the US nonsmokers (p = 0.001) but the dependence of CE on age was the same as for the US population (p = 0.043). Because the increase of MF of the Russians with age is 2-fold greater than that of the US smokers, the intensity of smoking and/or other environmental exposures, or the susceptibility to these exposures, must account for the difference in age dependent MF increase, not the proportion of Russians that are smokers. Differences in the lymphocyte subpopulations that survived the longer transit from Russia may have contributed to the observed differences in MF. However, overall, the mutant frequency results suggest that the Russians were chronically exposed to higher levels of agents that induce somatic mutation and that, on an age adjusted basis, the Russia population studied is at increased risk for health consequences from accumulated genetic damage.

Deletions and point mutations of mitochondrial DNA (mtDNA), which are characteristic of various human mitochondrial diseases, have been identified mainly in postmitotic tissues like brain, heart and skeletal muscle of healthy humans of advanced age but not in young people. An exponential increase with age was described for deletions of mtDNA. This paper reviews the molecular basis and experimental results on mutations of mtDNA in patients with mitochondrial diseases and in aged individuals. In addition new data on the exponential increase of point mutations of mtDNA, characteristic for MERRF and MELAS disease, in extraocular muscle from elderly humans are shown. Finally the ‘mitochondrial hypothesis on aging’ based on stochastic somatic mutations of mtDNA is presented.

Evidence is presented in support of the hypothesis that P DNA element movement in somatic cells of Drosophila melanogaster induces genetic damage that significantly reduces lifespan. The lifespan of D. melanogaster males was significantly reduced by the somatic movement of a single P element in the presence of P[ry⁺ Δ2–3](99B) transposase. In addition, the P[ry⁺ SalI](89D) repressor of P[ry⁺ Δ2–3](99B) somatic transposasc was observed to reduce the effect of P element movement on lifespan. Finally, the frequency of somatic-cell chromosome breakage was significantly increased in neuroblasts of males with somatically active P elements.

These results show that lifespan in D. melanogaster is decreased with increased somatic genetic damage from DNA-element movement. Although this conclusion does not confirm that transposable element movement is a cause of natural senescence, this conclusion is clear evidence in support of a close relationship between somatic genetic damage and aging.

The spontaneous frequencies of micronucleated reticulocytes (MNRETs) were examined monthly over the life spans of animals belonging to nine mouse strains for the 7th collaborative study organized by the CSGMT/JEMS MMS. Both sexes of the BDF1 strain and females of the A/J strain showed a statistically significant increase in mean spontaneous MNRET frequency in their last month of life, suggesting the possibility of strain-specific, age-dependent chromosomal instability. SAMP6/Tan, an accelerated senescence-prone strain, showed the same tendency, although it was not statistically significant. The other strains studied, ddY, CD-1, B6C3F1, SAMR1, and MS/Ac, did not show significant age-related differences in mean of MNRET frequencies. More extensive statistical analyses are underway, and the outcomes will be reported separately.

The level of spontaneous and gamma-radiation-induced mutations in the hypoxanthine-guanine phosphoribosyl-transferase (hprt) locus as well as the decrease in frequency of these mutations in mice of various age pretreated with dietary supplements of an antioxidant mixture (vitamins C, E, beta-carotene, rutin, selenium, zinc) were studied in splenocytes of young (8–14-week-old) and aged (102–110-week-old) male C57BL/6 mice.

The frequency of spontaneous mutations in splenocytes of 102–110-week-old mice was higher by 68–88% than that in mice aged 8–14 weeks. On gamma-irradiation (0.5–5.0 Gy) of mice, the frequency of radiation-induced mutations (Vf assay) in aged mice was 2.3 to 3.6 times (depending on dose) higher than in young ones.

Daily supplements of an antioxidant mixture to the diet of mice prior to irradiation showed an antimutagenic effect. The values of mutant frequency reduction factor (MFRF) for 14–110-week-old mice fed with dietary antioxidants during 6 weeks prior to gamma-irradiation with doses of 2.0 and 5.0 Gy were 5.4 and 3.7, respectively. The frequency of radiation-induced mutations prevented or not prevented by antioxidants was much higher in aged mice than in young ones.

Spontaneous mutant frequency of lacZ gene in spleen of transgenic Muta^™ mouse was examined at different ages. It was (3.2 ± 1.3 (SD)) × 10⁻⁵ at newborn and increased almost linearly with age up to (8.3 ± 1.8) × 10⁻⁵ at one year. Since the mutation of the gene is not likely to be subject to selection in vivo, the data support the idea that spontaneous mutation takes place throughout aging process and accumulates with age if not selected out by cell death.

The factors that influence the spontaneous mutant frequencies in mammalian tissues have been ranked on the basis of data from our laboratory together with published data. Some of the data come from the endogenous hprt and Dlb-1 loci, but most come from transgenic mice carrying the bacterial lad and lacI genes in recoverable lambda phage vectors. Since there is evidence that these bacterial loci are selectively neutral, the mutant frequency observed is the integral of the mutation rates from the formation of the zygote. The factors that affect the inferred mutation rate, in decreasing order of importance are: site of integration of the transgene, age, tissue, and strain. Insufficient data exist to determine the influence of gender (probably small) and inter-laboratory variables (probably at least as important as age). The two most surprising results arc (1) that about half of all mutations arise during development (and half of these in utero) and (2) that most somatic tissues, whether queiscent or actively proliferating, have similar mutant frequencies and similar increases during adult life.