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The Journals of Gerontology Series A: Biological Sciences and Medical Sciences 61:763-772 (2006)
© 2006 The Gerontological Society of America

Correlation Between the Level of Cytogenetic Aberrations in Cultured Human Lymphocytes and the Age and Gender of Donors

Alina Wojda, Ewa Zietkiewicz, Malgorzata Mossakowska, Wlodzimierz Pawlowski, Krzysztof Skrzypczak and Michal Witt

1 Institute of Human Genetics, Polish Academy of Sciences, Poznan, Poland.
2 International Institute of Molecular and Cell Biology, Warsaw, Poland.
3 Regional Hospital, Poznan, Poland.
4 Dispensary UNIMEDYK, Poznan, Poland.

Address correspondence to Michal Witt, MD, PhD, Institute of Human Genetics, Clinical and Molecular Genetics, Strzeszynska 32, Poznan, A 60-479, Poland. E-mail: wittmich{at}man.poznan.pl


   Abstract
Top
 Abstract
Materials and Methods
 Results
 Discussion
 References
 
To answer whether the age-related accumulation of chromosomal damage differs in men and women, and whether the aberration level in centenarians is proportional to their age, cytogenetic aberrations in dividing cells were analyzed. G-band karyotyping of mitotic spreads from lymphocytes was performed in 52 Polish centenarians and 71 controls (aged 21–78). Statistical evaluation was performed using nonparametric tests and regression analysis. The average level of all chromosomal aberrations was comparable in centenarians of both genders, but the age-related increase in chromosomal damage occurred faster in women than in men. Aging in both genders was marked by the increasing level of all aberrations rather than by chromosome-specific changes; the loss of X chromosome was the leading contributor in women. The age-related increase in the level of chromosomal damage reflected accumulation of dividing cells with a small number of aberrations. Individuals who survive to the extreme old age appear to accumulate aberrations at the slower rate.

AGING is a process that occurs in all members of a species. Typically conceived as characterizing a postreproductive period of life, this process in fact affects organisms of any age. It is the rate and accumulation of aging effects, which distinguishes young and old individuals. Aging in humans appears to result from a combination of genetically programmed phenomena and harmful environmental factors, both exogenous and endogenous.

These factors primarily act at the cellular level, rather than at the level of the organism (1,2). Cellular aging, termed "replicative senescence," involves irreversible inhibition of DNA replication and occurs in all eukaryotic cells. It is determined by telomere length, thus limiting cellular proliferation and growth after a certain number of cell divisions occurs ("Hayflick limit"). The ability of nondividing cells to maintain their metabolic activity leads to the accumulation of senescent cells in the organism (1,3–16). Because of the increase in the number of DNA lesions that accumulate during multiple repetitions of DNA replication throughout the proliferative life span of a cell, the accumulation of senescent cells in elderly persons might contribute to the aging of tissue and organism (2,17).

Cytogenetically, cellular aging is associated with a number of gross cellular changes, including cell-cycle arrest, increased and/or heterogeneous cell size, and a higher frequency of cells with various chromosomal aberrations. Classical and molecular cytogenetic studies of metaphases, interphase nuclei, and micronuclei in presenescent cells from elderly people, showed an increase in the frequency of cytogenetic changes. Studies of the genetic disorders characterized by the premature aging led to similar conclusions [e.g., (18)]. Although it is well recognized that cytogenetic analyses may provide a useful indicator of accumulated damage resulting from age and demonstrate how individuals differ in their ability to cope with cellular senescence, some discrepancies in assessing the role of cytogenetic aberrations as biomarkers of aging remain.

Several studies have shown a significant increase in chromosome loss (hypoploidy), primarily of the sex chromosomes, in peripheral blood lymphocytes and skin fibroblasts, in both men and women of advanced age. Some authors (19) found that the levels of chromosome-specific aneuploidy increased with the donor's advancing age, but others questioned the correlation between the level of autosome aneuploidies and age or sex (20–22).

Guttenbach and colleagues (22) demonstrated that sex-chromosome loss in women increased significantly only beyond the reproductive age; at the same time, the incidence of Y chromosome hypoploidy in men was shown to be distinctly lower than that determined for the X in women. Studies of metaphases showed that chromosomes most frequently involved in hypoploidies were the X in women and the Y in men (23,24).

Some studies reported significant age-related increase in the frequency of translocations, insertions, dicentrics, and acentrics (1) and of stable chromosomal aberrations in lymphocytes (25). The increased level of stable chromosomal aberrations (which accumulate at a significantly higher rate than does the unstable damage), is considered a reliable biomarker of aging in humans [for review, see (26)]. Generally, however, the data on the age-related changes in the amount and role of gaps and breaks and structural aberrations of chromosomes are limited; similarly, little is known about the correlation between age and polyploidy.

Centenarians, a small fraction of population at the extreme of human longevity, deserve closer attention when the correlation between the level of cytogenetic aberrations and age is considered. One of the basic questions is whether the genome stability in centenarians is better maintained in that group compared to the rest of the population. Because of a sparse number of centenarians available for the research, there are no extensive data available to date that would allow comparing the level of cytogenetic abnormalities in cells from centenarian donors and from younger controls.

In this study, we performed an extended cytogenetic analysis of dividing peripheral blood lymphocytes in a group of 52 unrelated Polish centenarians and in a control group of 71 individuals ranging from 21 to 78 years old (yo) stratified by age and gender. The aims were to (i) compare the level and profile of cytogenetic aberrations in these groups (ii) to answer whether the increase in the level of cytogenetic aberrations in centenarians is proportional to their old age and whether there are any gender-related differences in the cytogenetic aging process.


   MATERIALS AND METHODS
Top
 Abstract
 Materials and Methods
Results
 Discussion
 References
 
Heparinized blood samples were obtained, with informed consent, from unrelated individuals representing a random sample of the Polish population, men and women, stratified according to their age: 52 centenarians (46 women 100–108 yo, 6 men 100–102 yo), 20 individuals (9 women and 11 men) ranging in age from 21 to 30 yo, 20 (9 and 11) 40–50 yo, 13 (6 and 7) 60–68 yo, and 18 (8 and 10) 69–78 yo. All participants (centenarians and controls) were Caucasians of Polish origin, representing thus an ethnically homogeneous cohort. Age was accepted as reported by the participants in the study; reliability of the age claims in the group of centenarians was confirmed by inspection of relevant documents during blood collection. The controls were recruited at random at the regional diagnostic laboratory during yearly check-up procedures. All volunteers signed an informed consent.

Lymphocyte cultures were established from 0.3 mL of whole blood; blood was added to 4 mL of Eagle's medium (Biomed, Lublin, Poland) supplemented with 15% heat-inactivated fetal calf serum (Biomed), 1% penicillin solution (at 50 U/mL; Polfa, Tarchomin, Poland), and phytohemaglutinin (LF-7; Biomed), and was incubated at 37°C. After 71-hour incubation, Colcemid (Gibco BRL, Paisley, Scotland) was added to the medium (0.1 µg/mL). Lymphocytes were harvested 1 hour later by centrifugation (10 minutes at 150 g), resuspended in 0.075 M KCl, incubated for 20 minutes at 37°C, and fixed by gently mixing three times with methanol/acetic acid (3:1) for 20 minutes at 4°C. Fixed lymphocytes were applied onto clean glass slides and air-dried. Each karyotype typically records data from analysis of 20 G-banded mitotic spreads. The karyotype data sheets were reviewed to identify hypoploid, hyperploid, and polyploid cells, and structural aberrations of chromosomes. Typically {approx}200 (150–260) cells were analyzed for the presence of polyploidy.

The average level of various types of cytogenetic aberrations per cell (further referred to as the aberration level) was calculated as the number of aberrations divided by the number of metaphase spreads analyzed; the proportion of cells with aberrations was assessed in individuals or in groups, as indicated. Statistical tests (Kruskal–Wallis analysis of variance [ANOVA] by ranks and Friedman ANOVA, implemented using the Statistica package 6.0, StatSoft, Krakow, Poland) were used to determine the significance of the effect of independent variables on the level of aberrations in different age and/or gender groups. Because of the unequal sample size and unequal variance in the analyzed age and/or gender groups, the raw data (average number of aberrations per cell) were subjected to square root transformation [x' = sqrt(x) + sqrt(x + 1)] prior to the analysis; the transformed data fulfilled the required assumption of variance homogeneity (p >.1 in Levene's test, Statistica package). Regression analysis (Statistica package) was performed on the square root–transformed data to assess the slope and significance of the correlation of the aberrations level with age.


   RESULTS
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 Abstract
 Materials and Methods
 Results
Discussion
 References
 
The karyotypes of all examined individuals were normal (46,XX or 46,XY), with the only exception of a centenarian woman {46,XX,t(7;12)(q11.3;q14); this translocation was not included in further analyses)} (27). However, a certain fraction of mitotic spreads (of which typically 20 were analyzed per individual) contained a number of various chromosomal aberrations in all the groups analyzed. They included: (i) aberrations of chromosomal number (aneuploidies) and structure (chromosome gaps and breaks, translocations, deletions, other structural aberrations, Figure 1A), and (ii) polyploidies (Figure 1B). Except for few sporadic cases (10 mitotic spreads per total number of >2500 spreads analyzed), no aberrations in both chromosomes of a pair were observed. The data on sample size (number of probands, analyzed mitotic spreads) and the number chromosomal aberrations per cell stratified by age and/or gender groups are given in Table 1 (the detailed list of different types of aberrations in each analyzed individual is available from the authors upon request).


Figure 01
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  		Figure 1. Example of GTG karyotyping. A, Partial karyotypes showing different types of structural aberrations observed in lymphocytes from centenarians or controls. B, Karyotype with polyploidy

 

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  		Table 1. Incidence of Chromosomal Aberrations in Gender and Age Groups Examined.

 
Overall Levels of Chromosomal Aberrations
The overall level of all chromosomal aberrations (i.e., of aneuploidies, gaps and breaks and structural abnormalities considered together; polyploidy will be discussed in a separate section) appeared to be both gender-related and age-related. In the nonparametric test (Kruskal–Wallis ANOVA by ranks), the aberration level was significantly higher in women than in men (Hdf=1,N=123 = 14.7; p =.0001), and the increase in the level of aberrations with age was reflected by statistically significant differences between the age groups in both genders (Figure 2; men p =.0001, women p =.0034). In three younger groups (25–68 yo), the level of aberrations was significantly (almost 3 times) higher in women than in men (p <.001); in two older groups (>68 yo), the gender differences in the aberration level were not significant (p >.10), reflecting a plateau in the aberration level increase observed in women more than 60 yo.


Figure 02
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  		Figure 2. Age-related differences in the average level of all chromosomal aberrations in men and women. Aberrations per cell are expressed as the total number of aneuploidies, gaps/breaks, and various structural aberrations, divided by the number of mitotic spreads analyzed in each age and/or gender group. Age groups were significantly different as shown by Kruskal–Wallis analysis of variance (ANOVA) by ranks test: both genders together [Hdf=4,N=123 = 43.81, p <.000001; histograms not shown]; men [Hdf=4,N=42 = 24,04, p =.0001]; women [Hdf=4,N=81 = 15.72, p =.0034]

 
Contribution of different aberration types to the total level of chromosomal damage within each gender was significantly different as shown by a nonparametric Friedman ANOVA (men: Xdf=5,N=42 = 54.2, p <.000001; women: Xdf=5,N=81 = 238.6, p <.000001); p values remained significant also when only five types of aberrations (i.e., excluding hypoploidy) were compared (p <.010 in men and p <.00001 in women). Hypoploidy (chromosome loss) was the dominant type of aberration; the more detailed analysis of its accumulation with age is presented in the following section.

Hyperploidy (gain of an extra chromosome) and unstable structural changes (dicentric or acentric fragments, deletions, inversions, or isochromosomes) were observed only sporadically, and their accumulation with age will be not discussed. Spontaneous chromosomal gaps and breaks and stable cytogenetic damage (translocations, some deletions) were the most frequent aberrations after chromosome loss. Because the observed trends of positive correlation between stable structural rearrangements and age were not significant, only gaps/breaks will be discussed further in more details.

Hypoploidy
Depending on the age group, hypoploidy represented 45%–90% of aberrations in men and 55%–94% in women. Hypoploidy was the only type of aberration, the level of which (2 times higher in women) differed significantly between the genders in the total age range (Kruskal–Wallis ANOVA by ranks, p <.00001). This gender difference was mostly due to the contribution of the two youngest groups (21–50 yo), in which the average hypoploidy level in women was significantly higher than in men (Kruskal–Wallis ANOVA by ranks, p =.0001); in contrast, the hypoploidy levels in older groups (>60 yo) did not differ between the genders (p =.85). The differences in the level of hypoploidy in different age groups (Figure 3A) were significant in men (p =.0001) but not in women.


Figure 03
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  		Figure 3. Age-related changes in the level of hypoploidy in men and women. A, Levels of hypoploidy in different age groups in men and women. Age groups in men were significantly different as shown by Kruskal–Wallis analysis of variance (ANOVA) by ranks test [Hdf=4,N=42 = 24.83, p =.0001]; in women the age differences were not significant [Hdf=4,N=81 = 7.39, p =.1168]. B and C, Linear regression analysis demonstrating gender differences in the correlation of the level of autosomal (B) and sex chromosome (C) hypoploidy with age. The number of all autosomal losses or of sex chromosome losses divided by the number of mitotic spreads analyzed in each individual, after the square root transformation, were plotted against the donor's age. Statistics characterizing linear fits are shown under the graphs. NS = not significant; MS = marginally significant

 
To shed more light on the observed age and/or gender differences, we analyzed the level of hypoploidies stratified by chromosomes. In all the age groups pooled together, the X chromosome hypoploidy in women was several times higher than that of any other particular autosome (and of the Y chromosome in men). However, the total level of autosomal hypoploidies (i.e., the loss of all the autosomes considered together) was {approx}4 times higher than the level of female X chromosome loss.

Linear regression analysis (on square root–transformed data) revealed a highly significant positive linear correlation between the level of total autosomal loss and age in men (Figure 3B, p =.000003). The level of autosomal hypoploidy in women, although high, was not significantly correlated with age (p =.073). In men, the age-related increase in autosomal loss was steeper than in women, and the change in age explained more of the variance in autosomal loss (compare slope and r2 differences in Figure 3B).

In women, the only significant positive correlation of the chromosome-specific loss with age (Figure 3C) was observed for the X chromosome (p =.00004). In men, the significant positive correlation of the Y chromosome loss with age (Figure 3C, p =.0003) was due to the increased level of the Y hypoploidy in the oldest age group (unlike in younger men, in centenarians it was {approx}3 times higher than that of the average autosome).

Spontaneous Gaps and Breaks
In both genders, a positive correlation of the level of gaps and breaks with age was observed only in noncentenarians (Figure 4); in men, these aberrations appeared earlier, but reached their highest level later than in women (the gender differences in the level of gap and breaks concentrated in the age bracket of 40–78 yo but, even in this range, were not significant by the Kruskal–Wallis ANOVA test). In centenarians, the level of gaps and breaks decreased and was comparable in both genders. The regression analysis of the square root–transformed data indicated that the increase in the frequency of gaps/breaks in noncentenarians was significant in both genders (p =.00004 and p =.0001 in women and men, respectively); the negative correlation with age in people more than 68 yo was significant only in women (p =.0000007).


Figure 04
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  		Figure 4. Age-related changes in the level of spontaneous gaps and breaks in men and women. The level of gaps and breaks per cell (the number of breaks and gaps divided by the number of mitotic spreads analyzed in each individual) were square root–transformed and plotted against the donor age. Statistically significant linear fits based on the noncentenarian samples indicate positive correlation with age; linear fit in women more than 60 yo (dotted line) indicates negative correlation with age. Statistics characterizing linear fits are shown under the graphs

 
Analysis of gaps and breaks in the per-chromosome setting indicated that, in all the age groups, chromosomes from groups E and F (17–20), although affected in women, were not affected in men. Another noticeable difference concerned involvement of chromosomes with a large block of pericentromeric heterochromatin and of the X, which in the 60–68 yo group was higher in women than in men. Generally, however, the age-related involvement of specific chromosomes in structural aberrations was scattered and did not follow any clear trend.

Involvement of Fragile Sites in Structural Aberrations
To see whether common fragile sites and centromeres in the genome, known to be preferentially involved in chromosomal rearrangements, were the main source of structural aberrations, localization of the breaks and structural rearrangements along the chromosomes was analyzed. Among the total of 131 different structural aberration points observed in this study, 18 were at centromeres, 59 at noncentromeric common fragile sites, and 54 at other points of the chromosomes. In individuals more than 60 yo, the contribution of common fragile sites (centromeric and noncentromeric) to the overall number of structural aberrations (gaps/breaks, translocations, deletions, and other) per cell was 2–4 times higher than that of other points (Figure 5). No centromeric or noncentromeric fragile break points were seen in the youngest age group (21–30 yo) of both genders; it is interesting that centromeric fragile points were also not observed in 60–68 yo men. Centromeric break points occurred mostly in 60–78 yo women and in 69–78 yo men.


Figure 05
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  		Figure 5. Contribution of different break points to the overall amount of structural aberrations (gaps and breaks, translocations, deletions, and unstable aberrations) in each gender and age group

 
Distribution of Aberrations Among Mitoses
The observed age-related increase in the overall level of all chromosomal aberrations could be explained (i) by the increased proportion of metaphases with low numbers of aberrations in donors from older groups or (ii) by the increased frequency of the dividing cells, which accumulated large number of aberrations. To elucidate this question, we analyzed the correlation between age and the frequency of metaphases carrying different number of all chromosomal abnormalities (aneuploidies, gaps/breaks, and structural aberrations).

Square root–transformed frequency of metaphases carrying 1–3 and ≥4 aberrations (i.e., arbitrarily divided to represent "low" and "high" level of aberrations per cell) were plotted against the age of donors and subjected to regression analysis (Figure 6). When mitoses carrying small number of aberrations were analyzed, a significant positive correlation was observed within the whole age range in both genders (p =.0007 in women and p =.0000003 in men). In women, the positive correlation was even more significant in the group of noncentenarians (p =.0000002), followed by the plateau in the age bracket of 60–108 yo. Considering mitoses carrying large number of aberrations (≥4), the correlation was less or not significant and there was no plateau effect differentiating women from men. Thus, the age-related increase in the overall level of chromosomal abnormalities (as shown in Figure 2B) was due to the increasing incidence of cells containing small number of changes rather than to the accumulation of a large number of changes in few cells.


Figure 06
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  		Figure 6. The age-related changes in the frequency of mitoses carrying "small" and "large" numbers of aberrations in men and women. Square root–transformed frequencies of mitoses carrying small (1–3) and large (4+) number of aberrations (the number of all chromosomal aberrations, divided by the number of mitotic spreads analyzed in each individual) plotted against the donor age. Practically, the same results were obtained when mitoses were grouped to carry 1–2 versus >3 or 1–4 versus >5 aberrations (not shown). Linear fits are based on all samples. For mitoses carrying small number of aberrations in women, the fit based on the noncentenarian samples only is shown (dotted line). Statistics characterizing linear fits are shown under the graphs; NS = not significant

 
Polyploidy
Polyploid human cells observed in this study (Figure 1B) contained a duplicated copy of the diploid genome (92, sporadically less than 92 chromosomes). In younger groups, the levels of polyploidy were higher in women than in men, whereas in the oldest noncentenarians (69–78 yo) it was 4 times higher in men than in women (Figure 7). The significant positive correlation with age was observed in noncentenarian men (p =.0008) and in women more than 60 yo (p =.00007).


Figure 07
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  		Figure 7. Correlation of the level of polyploid cells and age in men and women. The average frequency of polyploid cells (the number of polyploidies divided by the number of mitotic spreads analyzed in each individual; on average, 150–250 mitoses were analyzed per individual) plotted against the donor age. Linear fits based on the noncentenarian samples and samples >60 yo are indicated by solid and dotted lines, respectively. Statistics characterizing linear fits are shown under the graphs. NS = not significant

 

   DISCUSSION
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 Abstract
 Materials and Methods
 Results
 Discussion
References
 
Both classical and molecular cytogenetic studies suggest that cellular aging is associated with a decrease in genome stability (1,3,8,13). In our study we expected that a profound comparative cytogenetic analysis of cells obtained from people of different ages may shed light on how chromosomal damage accumulates in aging cells and how individuals differ in their ability to cope with cellular senescence. Our observations confirmed that, indeed, there was an age-related increase in the number of cytogenetically identifiable abnormalities; the dynamics of these age-related changes differed in men and women, and depended on the type of aberration considered.

The overall number of all chromosomal aberrations per cell (including aneuploidies, gaps/breaks, and structural aberrations) was significantly higher in women than in men. This difference was noticeable already in the youngest age groups and most pronounced in the middle-age bracket (especially among individuals in their 60s). It is interesting that, in centenarians, the total level of cytogenetic aberrations was similar in both genders, reflecting the plateau effect observed in women more than 60 yo. In other words, subcentenarian women had more aberrations and acquired them earlier than did men, but those people who survived longer appeared to have similar aberration level in both genders.

The age-related increase in the level of sex chromosome losses is a well-documented phenomenon (20–24). Indeed, hypoploidy was the most frequently observed cytogenetic change in all the age-groups, systematically increasing in both genders throughout the whole age spectrum analyzed. The X chromosome loss in women was a predominant aberration, several times more frequent than that determined for the Y chromosome in men and for any single autosome. The higher incidence of the X chromosome loss in women, particularly pronounced in the metaphases from older participants, reflects a differential survival of various hypoploid cell types (20) consistent with the fact that X monosomies (e.g., Turner syndrome) exist in vivo (21,28), and indicates a lower importance of the redundant X for the survival of the cell and its decreasing role in older women. This explanation for the higher level of the X chromosome loss in women is consistent with those of previous studies, which repeatedly demonstrated that the chromosome loss in aging women most frequently involved the X (23,24). Guttenbach and colleagues (22) reported that the level of X chromosome loss in women increased significantly with age only beyond reproductive age. In our study, the positive correlation between the X loss and age in women was statistically significant for the whole age spectrum analyzed.

Previous studies on the age-related increase of the Y chromosome loss were discordant (20,22,29–31). In our study, the level of Y chromosome loss in noncentenarians was low and comparable to that of an average autosome, and of the male X; the frequency of the Y hypoploidy was visibly higher only in centenarians, consistent with the diminishing role of the Y chromosome (harboring the genes related to spermatogenesis and sex determination) in the oldest old men. The only trend in a loss of autosomes worth mentioning is that smaller ones appeared to be lost more frequently than longer ones, especially in women.

The age-related increase in the levels of autosome-specific aneuploidy remains a subject of controversy (17–22); furthermore, there is no uniform view on whether the incidence of autosome loss is inverse to chromosome length (22,31,32). In our study, losses of all chromosomes were recorded. Although the level of hypoploidy of particular autosomes considered separately appears not to be correlated with age, the overall amount of autosome loss increases with age in both genders, presumably reflecting the overall accumulation of cytogenetic damage with age.

It has been reported that the frequency of stable chromosome aberrations in lymphocytes increases with the donor age (25) at a significantly higher rate than that of the unstable damage; the higher frequency of translocations compared to dicentric or acentric fragments, deletions, inversions, or isochromosomes is consistent with their greater stability through the cell division (33). In our study, gaps/breaks and translocations were the most frequent among aberrations of chromosome structure. The unstable structural aberrations occurred at a very low level, and they most probably represented newly arisen events.

Spontaneous gaps and breaks are considered to be critical primary lesions in the formation of structural chromosomal aberrations; they may be induced by exogenous agents but also can occur spontaneously during the cell cycle (34,35); gaps and breaks analyzed in our study represented spontaneous aberrations. Many of the translocations involving chromosomes 7 and 14 occur in utero (and presumably correspond to immunoglobulin and related gene rearrangements) (35); other rearrangements seem to accumulate progressively and may reflect exposure to environmental agents. In our study, only a single case of translocation involving chromosomes 7 and 14 was found in all the groups analyzed (27). Breaks in the frequently affected region 9q10–9q12, usually considered artifacts of culture (36), were observed only four times.

Gaps and breaks preferentially occur at common fragile sites. These evolutionary conserved, specific micro- and mini-satellite regions in the human genome appear to be an intrinsic part of the chromosomal structure and are thought to be present in all individuals. Involved in chromosomal rearrangements and foreign DNA integration, they are a primary cause of the genome instability and as such may contribute to aging (37,38). As expected, the majority of chromosomal gaps and breaks and of stable structural aberrations were within the common fragile sites. The break points of gaps and breaks and of translocations and deletions frequently overlapped. The relatively high contribution of break points at centromeres in women more than 60 yo and in men older than 70 yo (one fourth and one third of the aberrations, respectively, occurred at only 14% of the observed break points) is presumably due to the lower stability of the repetitive centromeric sequences during repeated rounds of replication and recombination in aging individuals.

Repair-deficient cells may accumulate more DNA damage, resulting in the age-related increase in the amount of chromosomal aberrations (1). However, in our study proportion of mitoses with the very high number of aberrations remained low in all the age groups of both genders, the majority of aberrant cells harboring at the maximum 1–3 aberrations. In other words, no age-related accumulation of cells with exceptionally high number of aberrations was noticed. However, one has to remember that this observation concerns presenescent cells capable of undergoing division. Mitoses analyzed in this study did not include nondividing senescent cells, occurrence of which has been shown to increase with age (13).

It is a well-known fact that centenarian women far outnumber centenarian men, consistent with the average life span in women being 78–90 years and in men 70–87 years (Polish Yearbook of Statistics, Central Statistical Office, Warsaw, 2005). Recruiting in our study only 6 centenarian men compared to 46 women illustrates this discrepancy. Results of our cytogenetic analysis indicate that the level of genomic instability in centenarian men and women is similar. It is possible that they all recruit from among people whose genome instability level increases at a slower rate. According to the survival data, one could expect that women would be those participants with the lower level of aberrations. However, our data from the control groups (especially among individuals in their 60s and 70s) clearly indicate that women have more cytogenetic abnormalities of all types and that they acquire them earlier. This finding seems to suggest that women live longer despite the larger accumulation of cytogenetic aberrations during the earlier life phases. A number of possible explanations of this observation are considered below.

It has been reported that, in men, telomeres are shorter and cells divide faster than in women (14). It is therefore possible that inhibition of replication and accumulation of nondividing cells in men occurs at an earlier age than in women. The increasing proportion of nondividing cells in aging men could be one of the reasons why chromosomal aberrations (which normally arise during replications) do not accumulate at the same rate as they do in women.

Polyploidy is a relatively common event in eukaryotic organisms. It can arise by cell fusion, endoreduplication, or an abortive cell cycle; is associated with an orchestrated change in expression of several genes; and increases the probability of cell death (39–43). In our study, the frequency of polyploid cells in men and in women was similar in all age groups except for the oldest noncentenarians (69–78 yo), in whom it was 7 times higher in men than in women. It is interesting that this age group is characterized by the highest mortality rate in men. Polyploid cells arise during a variety of pathological conditions (e.g., myocardial hypertrophy and heart failure—a frequent cause of death in men; 44). Perhaps, the lower level of polyploidy in women than in men in their 70s contributes to a fact that women survive this age and more frequently live to become centenarians, despite the higher amount of aneuploidies and structural aberrations.

One of the hypotheses states that, considering the quality of centenarians' organism and cells, they are in fact in much the same position as is the normal middle-aged population (45). Our data suggest that this might be true indeed, at least for the part of population characterized by higher genome stability. The relatively low level of chromosomal abnormalities in the oldest old would reflect the slower rate of accumulation of genetic damage throughout their lives. In women, the high level of aberrations seen in the age group of 60–70 yo does not further increase in centenarians, indicating that there is a certain threshold of the genome instability that cannot be surpassed; those who survive, may therefore represent a subgroup characterized by the slower rate of abnormalities accumulation (comparable to the rate that characterizes men).

Summary
Although this article does not allow us to draw firm conclusions concerning the role of cytogenetic aberrations in survival, it does confirm that their level may serve as a biomarker of aging. The overall level of chromosomal aberrations, expressed as the average amount of the total chromosomal damage per cell, increases with the increasing age in both genders. Noncentenarian women in their 60s and 70s have more cytogenetic abnormalities of all types suggesting that during earlier life phases they accumulate more cytogenetic aberrations than do men. The level of genomic instability is similar in centenarian men and women, indicating that they all may recruit from among people whose genome instability increases at a slower rate. However, no predictive cytogenetic marker, indicating the higher chance of surviving into the very old age, could be identified. There are most likely many factors that play a role in the aging process, and there is no simple answer to the complex questions involving survival into extremely advanced age.


   Acknowledgments
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 Abstract
 Materials and Methods
 Results
 Discussion
 References
 
The data presented here were obtained within project PBZ-KBN-22/PO5/10 funded by the State Committee for Scientific Research (KBN) and coordinated by the International Institute of Molecular and Cell Biology in Warsaw.


   Footnotes
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 Materials and Methods
 Results
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 References
 
Decision Editor: James R. Smith, PhD

Received January 31, 2005

Accepted January 12, 2006


   References
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 Discussion
 References
 

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