Mutation Research/DNAging最新文献

Previous biochemical studies on pooled hepatocytes have provided a wealth of information concerning age-related changes in DNA damage and DNA vulnerability (susceptibility) to oxygen radicals and related oxidants, but these studies focused on the whole liver and not on individual hepatocytes. The present study was designed to clarify the DNA damage and DNA vulnerability to hydrogen peroxide in individual hepatocytes using single cell gel electrophoresis (comet assay), a method that measures DNA single-stranded breaks/alkali-labile sites in individual cells. Hepatocytes were prepared from the liver of young (6–11 months) and old (26–29 months) male Fischer 344 rats. DNA damage was induced by exposure to hydrogen peroxide (3 × 10⁻⁵, 3 × 10⁻⁴ and 3 × 10⁻³%). Observation of each comet image (migration length of DNA (MLD)) was performed in a non-exposure status (basal level) and after exposure. The mean value of MLD was significantly increased (approximately 1.5-fold) in the old rats at the basal level (P = 0.009). Moreover, the proportion of highly DNA-damaged hepatocytes (MLD > 80 μm) increased significantly (approximately 2.5-fold) in advanced age (P = 0.02). The mean value of MLD after exposure to hydrogen peroxide was increased with its concentration, but no significant difference was observed in DNA vulnerability to hydrogen peroxide between young and old rats. However, the proportion of hepatocytes showing a markedly high DNA vulnerability (MLD > 140 μm) to hydrogen peroxide was significantly higher in the old rats than in the young rats. It is suggested that the age-related increase in DNA vulnerability to oxygen radicals and/or related oxidants is some subpopulations causes the increase in DNA damage in advanced age in the liver as a whole.

We have measured DNa strand breaks induced by ionising radiation in nucleated cells from freshly isolated whole blood from normal human subjects. Samples werer taken after subjects had fasted overnight and again 1 h after they had eaten breakfast in combination with approximately 35 mg/kg vitamin C. Damage was measured by single cell gel electrophoresis (the ‘comet’ assay), in which DNA single strand breaks generate a comet tail streaming from the nucleus. In repeat experiments on 6 subjects a reduction in DNA damage, as indicated by a highly significant decrease in overall comet length, was observed following vitamin C ingestion, both in the unirradiated control blood samples and in the dose response to ionising radiation damage. In addition, consistent differences in dose response between individual subjects were found. The peak effect was 4 h after intake of food and vitamin C. An effect was also seen with vitamin C alone and after breakfast without additional vitamin C. Protection against strand breakage was also seen in Ficoll-separated mononucleasr cells but evidence was not obtained from protection of separated, mitogen stimulated T-lymphocytes either against ionising radiation cell killing in a clonal assay, or against clastogenicity assessed by micronucleus formation following one cell division. Exposure of separated lymphocytes in vitro to vitamin C, at doses greater than 200 μM, did not offer protection but induced strand breakage. Our results raise the possibility in normal diet may not only affect susceptibility to endogenous oxidative damage, but may affect some responses of the individual to radiation.

Cellular aging appears to be related to and perhaps caused by diminished DNA repair. To elucidate direct correlations between DNA repair capacity and senescence various parameters of cellular aging and DNA repair were studied simultaneously. Of special interest are features of DNA repair and senescence in cultured diploid fibroblasts derived either from healthy young or elderly probands as well as from patients suffering from premature senescence syndromes (Werner syndrome, Cockayne syndrome, ataxia telangiectasia and Down syndrome).

Here we demonstrate the striking parallelism between reduced maximal lifespan, elevated levels of spontaneous chromosomal breaks, higher incidence of formation of micronuclei, a significant prolongation cell cycle duration and a diminished reactivation of in vitro injured plasmid after transfection in cells from old individuals and from patients with premature senescence syndromes, suggesting a causal relationship between senescence and DNA damage.

Normal human diploid fibroblasts (HDF) have a finite life span in vitro and have used as a model system for the study of in vivo aging. Little in known about how changes in gene expression may affect the immortalization of human fibroblasts. We looked for cDNA clones whose mRNAs were differentially expressed between mortal senescent SV40-transformed human fibroblasts (B-32) and the immortal counterparts (B-32F) derived from B-32 cells. We identified three cDNA isolates by subtractive differential hybridization with ³²P-labeled cDNA probes from B-32 cells and B-32F cells. Nucleotide sequence analysis of these cDNA clones revealed that they wer homologous to the human vimentin, a human mitochondrial gene and a human gene of unknown nature. Slot blot and Northern blot analyses demonstrated that the former two were preferentially expressed in senescent B-32 cells and the last one was less expressed in B-32F immortal cells.

When supercoiled DNA is incubated Fe(II) at pH 7 in the presence of hydrogen peroxide, the rate of nicking first increases with increasing H₂O₂ concentration to reach a maximum, then decreases and eventually increases agein. When 0.1 mM histidine is added at neutral pH at low H₂O₂ concentration (< 3 mM), it hinders the nicking of DNA; when it is added at high H₂O₂ concentrations (> 10 mM), it enhances the rate of nicking. When similar experiments are performed at slightly acidic pH (4.5) the biphasic behavior is maintained, independent of the presence of histidine. One can conclude that the protonation of imidazole (pK = 5.9) abolishes the capability of histidine to modulate the oxidative degradation of DNA. Results of electron spin resonance experiments suggest that at low H₂O₂ concentration, the protective effect of histidine could be the consequence of its capability to bind OH^. radicals.

The Tc1 element of the free-living nematode Caenorhabditis elegans is a well characterized transposon that is present in 30–500 copies per haploid genome, depending on the strain. Excision of Tc1 elements, which occurs readily in somatic tissues during larval development, has not previously been examined during aging of adult worms. We have identified a recently inserted Tc1 element in the KR1787 mutator strains of C. elegans and have found that Tc1 somatic excision at that site increases by more than 14-fold during the organism's lifespan.

The rate of micronuclei counted on lymphocyte cultures from five healthy female donors, 27–80 years old, increased with age. Using pXBR1 probe, specific for the alphoid DNA of the X chromosome, the presence of this chromosome was investigated by FISH (fluoroscence in sity hybridization) in both micronucleic and metaphases. Both X aneuploidy and frequency of X chromosome per micronuclei increased with age. However, this overinvolvement of X chromosome was not sufficient to explain the overall increase of micronuclei with age, suggesting that autosomes are also involved. Thus, the higher increase of X than autosome aneyploidy in lymphocytes may result from both an excess of X choromosome losses and a better survival of cells with a monoosomy X.

Cytogenetic data on cultured lymphocytes of the in utero exposed A-bomb survivors in the RERF Adult Health Study cohort have been analyzed using the G-banding technique to determine the frequency of aneuploid cells. The data consist of blood samples collected between 1985 and 1987 from 264 Hiroshima individuals for whom DS86 maternal uterine dose estimates are available: 124 proximally exposed (74 males and 50 females) with an estimated dose of 0.005 Sv or more, and 140 distally exposed (76 males and 64 females) with a dose estimate of 0 Sv, assuming the neutron relative biological effectiveness (RBE) of 10.

A main feature of aneuploidy was the aneuploid frequency in autosomes depended generally on chromosome length; aneuploidies were significantly more frequent in shorter chromosomes than in longer chromosomes. The frequency of aneuploidies also depended on type, with chromosome loss approximately five times more frequent than chromosome gain. However, chromosome 21, as well as the sex chromosomes, were notable in that aneuploidy was much more frequent for these chromosomes than would be predicted from a simple relationship with length. X chromosome aneuploidies were significantly more frequent in females than in males. There was no dependence of aneuploid frequencies on dose when measured 40 years after the exposure.

I-compounds are species-, tissue-, genotype-, gender-, and diet-dependent bulky DNA modifications whose levels increase with animal age. While a few of these DNA modifications represent oxidation products, the majority of I-compounds appear to be derived from as yet unidentified endogenous DNA-reactive intermediates other than reactive oxygen species. Circadian rhythms of certain I-compounds in rodent liver imply that levels of these DNA modifications are precisely regulated. Caloric restriction (CR), the currently most effective method available to retard aging and carcinogenesis, has been previously shown to elicit significant elevations of I-compound levels in tissue DNA from Brown-Norway (BN) and F-344 rats as compared to age-matched ad libitum fed (AL) animals. The present investigation has extended this work by examining liver and kidney DNA I-compound levels in three genotypes of rats (F-344, BN, and F-344 × BN) and two genotypes of mice (C57BL/6N and B6D2F1) under identical experimental conditions in order to determine whether correlations exist between I-compound levels, measured in middle-aged animals, and median lifespan. Levels of a number of liver and kidney I-compounds were found to display genotype- and diet-dependent, statistically significant positive linear correlations with median lifespan in both species. In particular, the longer-lived hybrid F-344 × BN rats and B6D2F1 mice tended to exhibit higher I-compound levels than the parent strains. CR enhanced I-compound levels substantially in both rats and mice. Thus, I-compounds, measured at middle age, reflected the functional capability (‘health’) of the organism at old age, suggesting their predictive value as biomarkers of aging. The positive linear correlations between levels of certain I-compounds (designated as type I) and lifespan suggest that these modifications may be functionally important and thus not represent endogenous DNA lesions (type II), whose levels would be expected to correlate inversely with lifespan.

While the life-extending and disease-modulating effects of caloric restriction (CR) are well documented in whole animal studies and in correlative experiments using cells taken from CR animals, very few studies have used cells in culture after their removal from the CR-fed animal. In using this in vivo → in vitro approach we have attempted to examine the proposition that the effects of CR can be transferred to individual cells by analyzing the cellular functions of proliferation and transformation, the activation of oncogenes, and the methylation of DNA as a function only of diet. Pancreatic acinar cells excised from CR-fed Brown-Norway rats and placed in rich medium showed different responses compared to cells from ad libitum (AL)-fed controls. CR had the effect of slowing growth rate and protecting against spontaneous and -methyl-N′-nitro-N-nitrosoguanidine(MNNG)-induced transformation over 14 passages of cells in culture. At the molecular level, cells from the CR animals showed reduced c-Ha-ras oncogene expression and mutation as well as reduced mutation of the p53 suppressor gene. CR also increased genomic methylation of ras DNA. We conclude that the effects of CR treatment of the animal are transferred to individual cells and note that these responses (decreased proliferation and transformation; depressed oncogene expression and mutation and decreased suppressor gene mutation; and increased oncogene methylation) are cellular and molecular analogs of in vivo weight loss, life extension, and carcinogenesis modulation, which are hallmarks of CR in the whole animal. The fact that these responses are seen generations after the cells are removed from the CR-treated animal indicates that CR causes a permanent predisposition of pancreatic acinar cells to these modulated responses and shows the value of the in vivo → in vitro protocol in studies that relate diet to cellular and molecular function.