The terminus of a DNA helix has been called its Achilles' heel. Thus to prevent possible incomplete replication and instability of the termini of linear DNA, eukaryotic chromosomes end in characteristic repetitive DNA sequences within specialized structures called telomeres. In immortal cells, loss of telomeric DNA due to degradation or incomplete replication is apparently balanced by telomere elongation, which may involve de novo synthesis of additional repeats by novel DNA polymerase called telomerase. Such a polymerase has been recently detected in HeLa cells. It has been proposed that the finite doubling capacity of normal mammalian cells is due to a loss of telomeric DNA and eventual deletion of essential sequences. In yeast, the est1 mutation causes gradual loss of telomeric DNA and eventual cell death mimicking senescence in higher eukaryotic cells. Here, we show that the amount and length of telomeric DNA in human fibroblasts does in fact decrease as a function of serial passage during ageing in vitro and possibly in vivo. It is not known whether this loss of DNA has a causal role in senescence.

Telomeres shorten during ageing of human fibroblasts.

The Holy Grail of gerontologists investigating cellular senescence is the mechanism responsible for the finite proliferative capacity of somatic cells. In 1973, Olovnikov proposed that cells lose a small amount of DNA following each round of replication due to the inability of DNA polymerase to fully replicate chromosome ends (telomeres) and that eventually a critical deletion causes cell death. Recent observations showing that telomeres of human somatic cells act as a mitotic clock, shortening with age both in vitro and in vivo in a replication dependent manner, support this theory's premise. In addition, since telomeres stabilize chromosome ends against recombination, their loss could explain the increased frequency of dicentric chromosomes observed in late passage (senescent) fibroblasts and provide a checkpoint for regulated cell cycle exit. Sperm telomeres are longer than somatic telomeres and are maintained with age, suggesting that germ line cells may express telomerase, the ribonucleoprotein enzyme known to maintain telomere length in immortal unicellular eukaryotes. As predicted, telomerase activity has been found in immortal, transformed human cells and tumour cell lines, but not in normal somatic cells. Telomerase activation may be a late, obligate event in immortalization since many transformed cells and tumour tissues have critically short telomeres. Thus, telomere length and telomerase activity appear to be markers of the replicative history and proliferative potential of cells; the intriguing possibility remains that telomere loss is a genetic time bomb and hence causally involved in cell senescence and immortalization.

Telomere loss: mitotic clock or genetic time bomb?

The purpose of these guidelines is to provide concise guidance on the planning, performing and interpretation of studies to monitor groups or individuals exposed to genotoxic agents. Most human carcinogens are genotoxic but not all genotoxic agents have been shown to be carcinogenic in humans. Although the main interest in these studies is due to the association of genotoxicity with carcinogenicity, there is also an inherent interest in monitoring human genotoxicity independently of cancer as an endpoint. The most often studied genotoxicity endpoints have been selected for inclusion in this document and they are structural and numerical chromosomal aberrations assessed using cytogenetic methods (classical chromosomal aberration analysis (CA), fluorescence in situ hybridisation (FISH), micronuclei (MN)); DNA damage (adducts, strand breaks, crosslinking, alkali-labile sites) assessed using bio-chemical/electrophoretic assays or sister chromatid exchanges (SCE); protein adducts; and hypoxanthine-guanine phosphoribosyltransferase (HPRT) mutations. The document does not consider germ cells or gene mutation assays other than HPRT or markers of oxidative stress, which have been applied on a more limited scale.

/pdf/ipcs-guidelines-for-the-monitoring-of-genotoxic-effects-of-4c9e0ebfnh.pdf

IPCS guidelines for the monitoring of genotoxic effects of carcinogens in humans

The telomere hypothesis of cellular aging proposes that loss of telomeric DNA (TTAGGG) from human chromosomes may ultimately cause cell-cycle exit during replicative senescence. Since lymphocytes have a limited replicative capacity and since blood cells were previously shown to lose telomeric DNA during aging in vivo, we wished to determine: (a) whether accelerated telomere loss is associated with the premature immunosenescence of lymphocytes in individuals with Down syndrome (DS) and (b) whether telomeric DNA is also lost during aging of lymphocytes in vitro. To investigate the effects of aging and trisomy 21 on telomere loss in vivo, genomic DNA was isolated from peripheral blood lymphocytes of 140 individuals (age 0-107 years), including 21 DS patients (age 0-45 years). Digestion with restriction enzymes HinfI and RsaI generated terminal restriction fragments (TRFs), which were detected by Southern analysis using a telomere-specific probe (32P-(C3TA2)3). The rate of telomere loss was calculated from the decrease in mean TRF length, as a function of donor age. DS patients showed a significantly higher rate of telomere loss with donor age (133 +/- 15 bp/year) compared with age-matched controls (41 +/- 7.7 bp/year) (P < .0005), suggesting that accelerated telomere loss is a biomarker of premature immunosenescence of DS patients and that it may play a role in this process. Telomere loss during aging in vitro was calculated for lymphocytes from four normal individuals, grown in culture for 10-30 population doublings. The rate of telomere loss was approximately 120 bp/cell doubling, comparable to that seen in other somatic cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Loss of telomeric DNA during aging of normal and trisomy 21 human lymphocytes

Safety Evaluation and Risk Assessment of the Herbicide Roundup and Its Active Ingredient, Glyphosate, for Humans

In order to assess the potential of cytogenetic determinations on peripheral blood lymphocytes as a means of monitoring human populations subject to low level occupational and environmental exposures to chemical mutagens and carcinogens, accurate baseline data are required. Accordingly, we have determined mean frequencies of chromosomal aberrations and of sister-chromatid exchanges, their variances, and the sources of this variance in a cohort of 353 healthy employees of the Brookhaven National Laboratory. A detailed protocol was adopted for blood sampling, lymphocyte culture, cytogenetic preparation and scoring in order to minimize variation from these potential sources. Scoring was divided between the Oak Ridge and the Brookhaven groups with duplicate scoring sufficient to evaluate and minimize the effect of any differences between laboratories or between individual scorers. In all, the data include 71,950 cells scored for chromosomal aberrations and 16,898 cells scored for sister-chromatid exchanges. The mean unadjusted frequency of sister-chromatid exchanges was 8.29 +/- 0.08/cell. As reported in other studies, cigarette smoking very significantly influenced sister-chromatid exchange frequencies; in our study the mean for smokers was 9.0 +/- 0.2, while that for non-smokers was 8.1 +/- 0.1/cell. The mean frequency was statistically higher in females than in males, regardless of smoking status. On the other hand, age of the subject did not significantly influence sister-chromatid exchange frequencies. Curiously, the subject's total white cell count did influence sister-chromatid exchange frequency. No other source of variation was found. The frequencies of chromosomal aberrations of all types were determined. The frequency of the most common unequivocal chromatid type, the chromatid deletion, was 0.81 +/- 0.05%, that of the most common unequivocal chromosome type, the dicentric, was 0.16 +/- 0.02%. No statistically significant influence was found of age or sex, nor of any other parameter tested, on the frequency of any chromosomal aberration type, with the single exception of long acentric fragments, often "supernumerary", believed to represent X chromosomes precociously separated at the centromere. Such fragments were significantly more frequent in samples from females than those from males, and showed a significant positive regression on age.

Chromosomal aberration and sister-chromatid exchange frequencies in peripheral blood lymphocytes of a large human population sample.

We have previously reported on a cytogenetic-epidemiological study of chromosomal aberration and sister-chromatid exchange (SCE) frequencies in peripheral blood lymphocytes (PBL) of a cohort of 353 healthy employees of the Brookhaven National Laboratory (Bender et al., 1988). This sample has now been increased in order to extend the age range represented and, incidentally, the representation of non-white subjects. In total, the data now include chromosomal aberration information from 108,950 cells and SCE information from 25,397 cells from 613 samples from 493 subjects. Neither the mean frequencies of any of the chromosomal aberration types nor the mean frequency of SCE have changed notably through the addition of the new subjects and samples. The mean age at sampling of the population is now 43.1 years with a range of from 1.1 to 83.7 years. However, we still find no significant relationship of the frequency of any conventional aberration category to age with the single exception of the dicentric chromosome, which now shows a positive regression (p = 0.001). The raw mean SCE frequencies show a statistically significant increase with subject age, but when cigarette smoking status is taken into account, no significant age relationship is found. As with the earlier samples, neither aberration nor SCE frequencies was influenced by race. Mean SCE frequencies, measured in non-smokers, were about 5% higher in females than males. Only one aberration category, "supernumerary acentric fragment", was significantly related to sex. This "aberration", known to constitute early centromere separation of an X chromosome, is much more common in females than in males and, in the females, increases significantly with increasing subject age.

Chromosomal aberration and sister-chromatid exchange frequencies in peripheral blood lymphocytes of a large human population sample. II. Extension of age range.

Sister-chromatid exchanges (SCE), phenomena of incompletely understood mechanism or significance, are often enumerated as a measure of genotoxicity, as their frequency is clearly elevated by exposure to many, if not most, chemical mutagens and carcinogens (Latt et al., 1984). However, SCE arise spontaneously, and in fairly large numbers per cell on average, in the absence of known genotoxin exposure. Statistical tests to determine whether SCE levels are elevated in a population or in an individual are based on underlying assumptions regard-

B.E. Pyatt

Papers

Chromosomal aberration and sister-chromatid exchange frequencies in peripheral blood lymphocytes of a large human population sample.

Chromosomal aberration and sister-chromatid exchange frequencies in peripheral blood lymphocytes of a large human population sample. II. Extension of age range.

On the distribution of spontaneous SCE in human peripheral blood lymphocytes.

On the distributions of spontaneous chromosomal aberrations in human peripheral blood lymphocytes in culture.

Influence of white blood cell count on SCE frequency in peripheral lymphocytes