Chromosomal damage and ageing: effect on micronuclei frequency in peripheral blood lymphocytes

Age and Ageing 1999; 28: 393 397 1999, British Geriatrics Society Chromosomal damage and ageing: effect on micronuclei frequency in peripheral blood lymphocytes CLAUDIA BOLOGNESI, CECILIA LANDO 1,ALESSANDRA FORNI 2,ELEONORA LANDINI, ROBERTO SCARPATO 3, LUCIA MIGLIORE 3,STEFANO BONASSI 1 Toxicological Evaluation Unit and 1 Dipartimento di Oncologia Ambientale, Istituto Nazionale per la Ricerca sul Cancro, Largo R. Benzi 10, 16132 Genova, Italy 2 Istituto Di Medicina Del Lavoro, Clinica Del Lavoro Luigi Devoto, Università degli Studi di Milano, Italy 3 Dipartimento di Scienze dell Uomo e dell Ambiente, Università degli Studi di Pisa, Italy Address correspondence to: S. Bonassi. Fax: (+39) 10 5600501. Email: bonassi@hp380.ist.unige.it Abstract Background: instability in the organization and expression of the genetic material has been hypothesized as the basic mechanism of ageing. Objective: to quantify the effect of ageing on chromosomal damage as measured by spontaneous micronuclei (MN) frequency in peripheral blood lymphocytes. Method: analysis of a large population sample from two laboratories applying the cytokinesis-block technique and a third using traditional interphase analysis. The age-related effect on baseline level of micronuclei frequency and on cell proliferation measures was further investigated in a study of peripheral blood samples from healthy subjects. Results: there was an increase of MN frequency with age. The regression lines showed a positive slope and were statistically significant (P < 0.01) with a steeper trend for cytochalasin B-treated samples. An inverse correlation with age was detected for the percentage of binucleated cells in laboratories using cytochalasin B. This study confirms the increase of basal level of MN with age. A decrease by age in proliferation efficiency measured by the percentage of binucleated cells suggests an interference of age-related factors on cell division. Conclusion: there is an increase in MN frequency with increasing age. Keywords: biomonitoring in human population, chromosomal damage and ageing, micronucleus test Introduction The ageing process consists of structural alterations and functional declines in body systems with a consequent impairment of homeostasis with increased vulnerability to age-related diseases, ultimately leading to death [1]. The effects of ageing in individuals appear to be a combination of genetically programmed processes [2] and genetic alterations induced by exogenous and endogenous factors [3]. The increase of cellular components, damaged by highly reactive free radicals [4 7] and associated with decreased DNA repair capability [8, 9], play a central role in genetic instability [10], a common marker of cancer and age-associated degenerative diseases [11]. There is an age-related increase in DNA damage and in the mutation rate [12, 13] and variations in different genetic end-points such as DNA adducts [14], DNA breaks or alkali-labile sites [15, 16], mutations at different genetic loci [17 19] and chromosomal damage [20, 21]. The presence of chromosomal abnormalities was the first genetic change to be specifically associated with age [22 24]. An increase in basal levels of chromosomal damage dependent on age has been detected for sister chromatid exchange, chromosomal aberrations and micronuclei (MN) frequency [25]. Age affects sister chromatid exchange, although the increase in old age is limited ( 10 15%). The effect of age on chromosomal aberration frequency is unclear. Recently, biomonitoring studies showed that stable cytogenetic changes accumulate with age [20, 21, 26]. Most studies have found an increase in MN frequency with age [25]. We have investigated the quantitative effect of ageing on MN frequency and associated biological indices by re-analysing a large 393

C. Bolognesi et al. Table 1. Characteristics of study group in the three laboratories % of subjects... Age Mean (SD)...... No. of With occupational Laboratory subjects Cells scored Male exposure Smokers Mean (SD) Range MN % BC... DSA-PI 206 486 000 81 62 36 40.2 (9.7) 20 59 7.0 (5.1) 30.1 (12.8) IST-GE 403 897 017 79 58 29 40.6 (10.7) 20 68 5.9 (3.6) 24.5 (12.2) MDL-MI 178 554 000 80 68 51 41.1 (11.8) 20 77 2.7 (1.9) BC, binucleated cells; MN, micronuclei. population sample from the Italian collaborative project MEDB (Molecular Epidemiology Data Bank) [27]. We also examined peripheral blood lymphocytes of healthy individuals. Materials and methods Population study A subset of 791 subjects investigated for MN frequency, between 1984 and 1994, in three laboratories were selected from the data bank of the MEDB project. MN frequency was evaluated in cultured cytokinesisblocked lymphocytes [28] (IST-GE and DSA-PI) and by traditional interphasic analysis (MDL-MI). (Further information on sampling, laboratory protocol and scoring procedures of the original studies is published elsewhere [25].) We obtained information on age, sex, occupational exposure, number of scored cells and, for IST-GE and DSA-PI, proportion of binucleated cells (Table 1). The subjects were aged from 20 to 77 years, and were divided into five age-groups. For the DSA-PI laboratory, some analyses were restricted to the age-band 50 59 years because of the small number (four) of subjects over 60. Further analyses of MN frequency were carried out using the standardized variable (smn) [29]. The association between age and the outcome variables was evaluated through univariate parametric analysis, using SPSS for Windows statistical software [30]. Experimental study Thirty healthy male volunteers, who were non-smokers were selected for the experimental study (Table 2). Blood samples were collected in sodium heparin tubes and coded before culturing. For each person, two parallel cultures were set up with 0.3 ml of whole blood added to 4.7 ml of HAM s F10 (ICN, Costa Mesa, CA, USA) supplemented with 10% foetal bovine serum (ICN, Costa Mesa, CA, USA), 1.5% phytohaemagglutinin (Murex Diagnostic, Dartford, UK). Cytochalasin B (Sigma Chemical Co., St Louis, MO, USA; final concentration 3 mg/ml) was added after 44 h to a single culture to block cytokinesis. These cultures were harvested at 72 h after the addition of cytochalasin B. Parallel cultures not treated with cytochalasin B were harvested at 36 and 72 h. At the end of incubation, microscopic slides were prepared [31]. In accordance with standard criteria [28], 2000 mononucleated or binucleated lymphocytes were scored for MN identification for each subject. Table 2. Frequency of micronuclei by age, presence of cytochalasin B (cyt-b) and culture time Mean micronuclei (and SD)... Mononucleated... Age-group Cyt-B 36 h 72 h Binucleated (72 h)... I( 19) 0.1 (0.2) 0.6 (0.7) a þ 0.3 (0.5) 0.9 (0.9) a II (20 44) 0.1 (0.3) 0.4 (0.5) þ 0.4 (0.4) a 1.4 (0.8) b III ( 45) 0.3 (0.5) 0.6 (0.8) þ 0.9 (0.8) 2.6 (1.7) b Total 0.2 (0.3) 0.6 (0.7) b þ 0.6 (0.6) b 1.6 (1.4) b Student t-test (reference value 36 h mononucleated): a P < 0.05; b P < 0.01. 394

Chromosomal damage and ageing The association between age-group and MN frequency was tested using parametric tests, after appropriate transformation of original values (arcsin x)to normalize data distribution. Results Population study The main characteristics of the three datasets evaluated in the population study are shown in Table 1. The distribution of the explanatory variables is homogeneous across laboratories, with 80% being males, the proportion of subjects occupationally exposed to mutagens ranging from 58 to 68%, and a mean age of about 40 years. Current smokers were more represented in the MDL-MI samples (51%). The frequency of micronucleated cells, as well as the proportion of binucleated cells, were comparable in the two laboratories using the cytokinesis-block technique, whereas the laboratory MDL-MI showed a considerably lower value. A clear increase of MN frequency with age was found in all samples (Figure 1). All regression lines showed a positive slope and were statistically significant (P < 0.01), although data from laboratories using cytochalasin B showed a somewhat steeper trend. The use of standardized MN frequencies allowed us to perform a pooled analysis of population data, and revealed a different trend by age-band in males and females. Men had a value of smn constantly lower than women, with statistically significant differences in the three central age-bands. Moreover, they showed a constant increase throughout age-bands, while women had a steeper increase of MN frequency Figure 2. Frequency of standardized micronuclei (smn) by age-group and sex. B, female; A, male. Student s t-test: n.s., not significant; *P < 0.01. during adult life, followed by decline in the oldest group (Figure 2). The association between the proportion of binucleated cells and age was evaluated in laboratories performing cytokinesis-block technique (Figure 3). Material from both the DSA-PI and IST-GE laboratories showed an inverse correlation with age in both sexes, i.e. b= ¹0.16 in men and b = ¹0.28 in women, both coefficients being statistically significant at the level of P < 0.01. Figure 1. Frequency of micronuclei by age-group and laboratory. Figure 3. Proportion (%) of binucleated cells (BC) by agegroup and sex. ---, female;, male. 395

C. Bolognesi et al. Figure 4. Proportion of binucleated cells (BC; transformed value) by age. Cells were treated with cytochalasin B, 72 h culture (30 subjects). Experimental study The average frequency of MN in mono- and binucleated lymphocytes at 36 h (mononucleated only) and 72 h (mono- and binucleated) in the three age-groups is shown in Table 2. An increased level of MN was observed in cells cultured for 72 h compared with the 36 h values. We did not find any influence of age on MN frequency in mononucleated lymphocytes harvested at 36 h (the total frequency being very low at 0.16 0.35 per thousand). The regression analysis of age on micronucleated lymphocytes frequency at 72 h in cytochalasin B-treated cells revealed a significant association in both mono- (P < 0.05) and, to a greater extent, binucleated cells (P < 0.01). The presence of association between age and the proportion of binucleated cells in 72-h cultures in presence of cytochalasin B was tested through linear regression analysis (Figure 4). The slope was negative as expected (b = 0.001; not significant), but failed to reach statistical significance. Discussion A clear association between age and MN frequency in peripheral blood lymphocytes, and an age-dependent variability of associated biological parameters was found in both population and experimental studies. Experimental factors such as culture time and use of cytochalasin B are important determinants in the evaluation of age-related effects on MN frequency. Mean frequencies of MN in both mononucleated and binucleated lymphocytes increase with harvesting time. The increase in the size of cell populations undergoing division raises the probability of more cells expressing damage. In our experimental studies, the basal level of MN is highest in binucleated lymphocytes harvested at 72 h and is blocked by cytochalasin B, a culture factor which increases assay sensitivity [28]. Much evidence of an age-related increase in basal MN frequency has come from biomonitoring studies [25] which have evaluated MN frequency in 72-h cultures. The comparison of three laboratories revealed a different degree of association between MN frequency and age (Figure 1). The use of cytochalasin B seems to have strengthened the relationship. In our study, ages under 20 are missing, while those over 60 are underrepresented. This does not alter our conclusions, since major changes in the trend of MN frequency by age should occur in the age span studied [25]. Furthermore, age-related factors could interfere with cell division, modifying the percentage of binucleated cells in lymphocytes harvested at 72 h when cells have already divided once. In keeping with this hypothesis, a decrease in binucleated cells with age was evident in both our population and experimental studies. This index has been used as a proliferation index of culture: in aged people, diminished frequency of binucleated lymphocytes could mean a slowing of proliferative capacity. An age-related decline in mitotic index associated with an increase of chromosomal abnormality and MN frequency occurs in both sexes [24]. A different proliferation activity may explain the discrepancy between sexes in the trend of MN frequency with age (Figure 2). Our evidence (Figure 3) confirm the reduction of cell proliferation in the oldest groups and the constantly higher proportion of binucleated cells in women. In contrast, the parallel trend in both sexes in the proportion of binucleated cells by age, excludes this factor as a possible cause of the age sex interaction. The increase in spontaneous chromosomal instability with age, as reflected in the higher basal level of MN frequency, is associated with an accumulation of DNA damage due to a progressive impairment of overall DNA repair capacity [15]. A similar pattern of chromosomal instability occurs in different premature ageing syndromes characterized by defective DNA damage defence mechanisms [11]. Changes in chromosomal structure or function are strongly associated with ageing, although our results cannot determine whether these changes are part of the cause or a consequence of ageing. Key points Ageing is associated with changes in chromosomal structure and function. Spontaneous micronuclei frequency in peripheral blood lymphocytes is a measure of chromosomal damage. There is an age-related increase of spontaneous micronuclei frequency in peripheral blood lymphocytes. 396

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