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Age-dependent changes in spontaneous frequency of micronucleated erythrocytes in bone marrow and DNA damage in peripheral blood of Swiss mice
Accepted Manuscript Title: Age-dependent changes in spontaneous frequency of micronucleated erythrocytes in bone marrow and DNA damage in peripheral blood of Swiss mice Author: Hari N. Bhilwade S. Jayakumar R.C. Chaubey PII: DOI: Reference:
S1383-5718(14)00160-0 http://dx.doi.org/doi:10.1016/j.mrgentox.2014.04.026 MUTGEN 402497
To appear in:
Mutation Research
Received date: Revised date: Accepted date:
20-8-2013 25-3-2014 30-4-2014
Please cite this article as: H.N. Bhilwade, S. Jayakumar, R.C. Chaubey, Agedependent changes in spontaneous frequency of micronucleated erythrocytes in bone marrow and DNA damage in peripheral blood of Swiss mice, Mutation Research/Genetic Toxicology and Environmental Mutagenesis (2014), http://dx.doi.org/10.1016/j.mrgentox.2014.04.026 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Highlights: 1. Ageing enhanced the bone‐marrow micronucleated erythrocytes in mice 2. As compared to males, females showed higher micronuclei frequency with age
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3. Enhancement of DNA damage was also observed with age in mice
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Age-dependent changes in spontaneous frequency of micronucleated
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Swiss mice
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erythrocytes in bone marrow and DNA damage in peripheral blood of
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Hari N.Bhilwade , S. Jayakumar, R.C.Chaubey Radiation Biology and Health Sciences Division, Bhabha Atomic Research Centre,
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Mumbai 400 085, India
*Corresponding author
Radiation Biology and Health Sciences Division Bhabha Atomic Research Centre Mumbai 400 085, India
Email: [email protected] Tel.: +91-22-2559-0425 FAX: +91-22- 2550-5151
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Abstract
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Age-dependent changes in chromosomal damage in bone marrow – a selfproliferating tissue – in the form of spontaneously occurring micronucleated erythrocytes,
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and DNA damage in peripheral blood were examined in male and female Swiss mice. In the erythrocyte population in the bone marrow, polychromatic (immature) erythrocytes
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showed a significant increase in the frequency of micronuclei as a function of age of the mice (1–20 months). The increase in micronucleus frequency was less in normochromatic
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(mature) erythrocytes. The female mice showed a higher frequency of micronuclei than
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the male mice in all the age groups examined. However, the female to male ratio of micronucleus frequencies in total erythrocytes as well as in polychromatic erythrocytes decreased with age. DNA damage, measured as tail moment in the single-cell gel electrophoresis in peripheral blood of different age groups of mice (1, 6, 12 and 18 months) showed a gradual increase with age. Female mice showed more DNA damage than 1-month and 18-months old male mice. In conclusion, these results show that there is an accumulation of genetic damage in bone marrow and DNA damage in peripheral blood of mice during ageing, and that females show more alterations than males.
Keywords: Ageing, DNA damage, micronucleus assay, comet assay
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1. Introduction Age is one of the important confounding factors that influence the response of an organism to physical, chemical and other environmental agents. Ageing is a natural
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process in which the functions of tissues and organs of the body diminish with advancing age. The exact mechanism of ageing is unclear, but the hypothesis that ‘ageing is a
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consequence of alterations of cellular macromolecules’ is supported by several studies at
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the cellular and tissue level of higher organisms [1]. DNA, the genetic material, is the primary object of studies related to the ageing process. The somatic mutation theory of
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ageing tries to explain the process as a function of genetic alterations in somatic cells, mainly involving DNA and deterioration of chromatin organization [1, 2]. In a number of
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studies, changes in chromosomal DNA are correlated with ageing, both in human and animal models [3], by use of various cytogenetic parameters such as chromosomal
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aberrations, sister chromatid exchange and micronuclei [4]. Using the micronucleus
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assay, Ganguly et al., demonstrated that the frequency of micronuclei in bi-nucleated cells of human peripheral lymphocytes increased with age [5]. On the contrary Fenech et al., observed no such age-dependent increase in micronucleus frequency in humans [6]. On the other hand, Orta & Gunebaken observed an increase in micronucleus frequency until the age of 50 and then a decrease up to the age of 73 in humans [7]. Besides these human studies, several investigations have been carried out in mouse models regarding changes in spontaneous frequency of micronuclei upon ageing [8-12]. Among these, some have shown an age-dependent increase in the micronucleus frequency in mice [8, 11]. However, in other studies with mice and rats, such an increase could not be observed [9, 10, 12]. Similar inconsistencies have also been reported with other cytogenetic
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endpoints in human and animal models, suggesting the need for further studies in this area, with a combination of different techniques. This will help build consensus on the correlation between ageing and different cytogenetic parameters. Besides, the
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information on changes in baseline frequency of cytogenetic endpoints with advancing age can be useful when comparing situations when baseline frequencies of exposed
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individuals are not known prior to accidental exposure [4]. Moreover, it would also help
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in comparing environmental risk or exposure in different age groups.
In the present study, we have combined the micronucleus test and the comet assay
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to study the spontaneous micronucleus frequency in bone marrow and the baseline level of DNA damage in peripheral blood, respectively, in different age groups of mice. The
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frequency of micronuclei is a reliable measure of both chromosome loss and breakage, but can only be conducted in dividing cells. On the other hand, the comet assay can detect
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a broad spectrum of DNA damage (including DNA single- and double-strand breaks,
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base damage, alkali-labile DNA adducts, and other DNA lesions associated with diverse reactive oxygen species) virtually in any tissue [13]. Since there is paucity of data on changes in incidence of micronuclei in the bone
marrow as well as DNA strand-breaks in mice as a function of age, this study was aimed to examine the frequency of spontaneously occurring micronucleated erythrocytes in bone marrow by means of the micronucleus test, and of DNA strand-breaks with the comet assay, in different age groups of mice. The role of sex on the incidence of chromosomal/DNA damage with advancing age has also been analyzed.
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2. Materials and methods 2.1 Materials Fetal calf serum (GIBCO, USA), May-Gruenwald and Giemsa stains, SYBR-
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green-II stain, low-melting agarose, ethylenediammine tetraacetic acid (Na2-EDTA), Triton X-100, dimethyl sulfoxide (DMSO), Tris HCl, (Sigma Chemicals, USA),
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methanol, xylene (Qualigens fine chemicals, Glaxo, Bombay, India), NaCl (SD Fine
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chemicals, India), NaOH (Thomas Baker Chemicals Ltd, India) and fully frosted slides (Erie Scientific, Sybron, Portsmouth, N.H. USA) were used in these studies.
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2.2 Animals
Inbred albino Swiss male and female mice reared in the animal house of Bhabha
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Atomic Research Centre, Mumbai were used in this study. All the animals were caged individually and maintained under controlled conditions of temperature (23-25°C), and a
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libitum.
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12h/12h light/dark cycle. Animals received nutritionally balanced food and water ad
2.3 Micronucleus test
Six to ten mice of either sex of different age groups (1, 12 and 20 months) were
used for the bone-marrow micronucleus assay. Animals were processed for enumerating micronuclei in the erythrocytes as described previously [14] with minor modifications as reported earlier [15]. In brief, bone marrow was flushed in a 5-ml centrifuge tube containing fetal bovine serum and centrifuged at 1500 rpm [give proper ‘x g’-value here; Ed.] for 5 min. The cell pellet was mixed thoroughly, bone-marrow smears were made on clean glass slides and stained as follows: 5-min incubation in undiluted MayGruenwald (0.25% in methanol), 3 min in diluted May–Gruenwald solution (1:1, May–
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Gruenwald/distilled water), rinsed in distilled water three times and then stained with diluted Giemsa (1:6 of the Giemsa stock/distilled water) for 10 min following [followed by? Ed.] thorough washing with distilled water. The slides were dried, cleared for 5 min
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in xylene, and mounted in DPX. Two slides were made per animal. Coded slides were scored for the incidence of micronucleated polychromatic erythrocytes (Mn-PCEs) and
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micronucleated normochromatic erythrocytes (Mn-NCEs) at 100x magnification under
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oil-immersion. At least 2000 polychromatic erythrocytes (PCEs) with or without micronuclei and a corresponding number of normochromatic erythrocytes (NCEs) were
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scored per animal. 2.4 Alkaline comet assay
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For detection of total DNA strand-breaks in peripheral blood leukocytes, the alkaline comet assay (single-cell gel electrophoresis) was used [16]. For this assay, about
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50 µl of heparinised whole blood from four mice (two males, two females) belonging to
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different age groups (1, 6, 12 and 18 months) were collected and mixed with 1.0 ml of 0.8% low-melting agarose solution prepared in 0.9% saline at 38ºC, and evenly layered on fully-frosted slides. After solidification of the agarose, the slides were kept in lysis buffer (2.5 M NaCl, 100 mM Na2-EDTA, 1% Triton X-100, 10 mM Tris-HCl [pH? Ed.] and 10% DMSO) for 1h at 4ºC. The slides were removed from the lysis solution, washed with alkaline buffer, and placed on a horizontal electrophoresis apparatus in alkaline buffer (300 mM NaOH, 1 mM Na2-EDTA, pH 13.0) at room temperature for 20 min to allow DNA unwinding and expression of alkali-labile sites. Electrophoresis was carried out for 20 minutes at 0.8V/cm. Afterwards, the slides were washed gently in neutralizing buffer (0.4 M Tris-HCl, pH 7.5) to remove alkali and detergents, stained with SYBR
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Green II dye and observed under a fluorescence microscope (Carl Zeiss Axio-vision) at 40x magnification. Images of 50-60 individual cells per slide were acquired with a digital imaging system and stored as bitmap file. These images were analyzed with SCGE-PRO
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software [17] and different parameters of DNA damage such as % DNA in tail (%DNAT), tail length (TL) and tail moment (TM, product of fraction of DNA in tail and tail
2.5. Measurement of Reactive Oxygen Species levels
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have used tail moment for representing DNA damage [18].
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length) were obtained. As the tail moment is considered as the optimum parameter, we
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Reactive oxygen species (ROS) levels in cytosol and mitochondria were measured with dichlorofluorescein and rhodamine123, respectively. For measurement of ROS,
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splenocytes were isolated from male Balb/C mice of different age: 1 month (N=4), 5 months (N=4), 10 months (N=2), 11 months (N=2), and 13 months (N=2), and 5x10
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cells were placed in 96-well plates and treated with either dichloro-dihydrofluorescein
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diacetate (DCFDA - 10µM) or dihydrorhodamine123 (DHR123 - 10µM). Thirty min after the treatment, fluorescence was read with a fluorimeter (DCF: λex 485nm and λem 535nm; Rhodamine123: λex 511nm and λem 535nm). 2.6. Statistical analysis
In the micronucleus test and the comet assay, means and standard deviations were
calculated based on the micronucleus frequency or the tail-moment values obtained from individual animals of the particular age group. Statistical significance between the groups was analysed by use of the t-test and analysis of variance (ANOVA), with GraphPad Prism 5.0. Values were considered significant at p < 0.05.
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3. Results 3.1. Micronucleus frequency increases with advancing age in mice The micronucleus assay was conducted in 1-, 12- and 20-month old Swiss mice,
dependent
increase
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micronucleus
frequency
both
in
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to determine whether the incidence of micronuclei varied with age. We observed an agepolychromatic
and
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normochromatic erythrocytes of the bone marrow. The frequencies of Mn-PCEs, Mn-
NCEs, and total micronucleated erythrocytes (Mn-Es) in the bone marrow of male and
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female mice in different age groups are summarized in Table 1.
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In male mice, a significant increase in the frequency of Mn-PCEs per thousand cells was observed at 12 months (2.81±0.74) and 20 months (3.22±0.82) compared with
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that of one-month-old mice (1.53 ±0.94). Similar to males, female mice also showed a significant increase in spontaneous frequency of Mn-PCEs upon ageing. The spontaneous
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frequency of Mn-PCEs in one-month-old female mice was 2.56±0.61, compared with
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3.71±0.69 at 12 months and 4.07±0.89 at 20 months (Table 1, Table 2). The frequency of Mn-NCEs and Mn-Es followed the same trend as Mn-PCEs, both in male and female mice. However, the frequency of micronuclei observed in NCEs was lower than that in PCEs in all age groups, both in male and female mice. When we compared the difference in micronucleus frequency between 12- and 20-months-old mice, Mn-PCEs, Mn-NCEs and Mn-Es did not change significantly. The PCEs/NCEs ratio, reflecting the proliferative activity of the bone-marrow cells, remained comparable between males and females of different age groups. Another interesting observation in this study was that the female mice showed a higher frequency of Mn-PCEs, Mn-NCEs and Mn-Es than the males, in all age groups (Table 1, Table 3). The frequency of Mn-PCEs in one-month-old female mice was
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2.56±0.61, which was significantly higher than the corresponding frequency of Mn-PCEs in one-month-old male mice (1.53±0.94). Similarly, at 12 months female mice showed a significantly higher frequency of Mn-PCEs than the male mice. However, at 20 months,
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although the female mice still showed a higher Mn-PCE frequency than the males, it was no longer significantly different. The ratio of Mn-Es frequency between female and male
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the frequencies at 12 months (1.29) and 20 months (1.14).
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was also analyzed (Fig.1) and at one month it was found to be highest (1.46), followed by
The slope depicting the mean spontaneous frequency of Mn-PCEs in male and
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females with increasing age was also calculated (Table 4). The rate of induction of MnPCEs was relatively high in the younger age groups (1-12 months) in both males and
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females compared with the older age groups (12-20 months) of either sex, which was reflected by a steeper slope. The slope for Mn-PCEs in males was higher than that of
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females in younger (1-12 months) as well as older (12-20 months) age groups.
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3.2. DNA damage increases with advancing age in mice DNA damage in terms of tail moment observed in male and female mice of
different age viz. 1, 6, 12 and 18 months, was measured by use of the alkaline comet assay (Fig. 2). Similar to the micronucleus frequency in the bone marrow, the spontaneous DNA damage also showed a statistically significant, age-dependent increase in male mice. The tail moment observed in one-month-old male mice was 1.33±0.16, which had increased more than two-fold at six months (3.17±0.12), and it continued to increase up to 18 months. But the rate of increase in tail moment was comparatively slow between 12 and 18 months. Likewise, in females a similar age-dependent increase in spontaneous DNA damage was observed, as reflected in tail-moment values in the comet
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assay. The statistical parameters characterizing the DNA-damage response (tail moment) with age in male and female mice were calculated (Fig.3). In the age-dependent increase in DNA damage, the males showed a steeper slope (0.153) than the females (0.121).
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3.3. Levels of reactive oxygen species (ROS) show age-dependent increase in mice ROS levels are a good measure of oxidative stress in the cell. We have measured
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the cytosolic and mitochondrial ROS levels in splenocytes of mice belonging to different
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age groups (1, 5, 10, 11 and 13 months) by measuring the fluorescence of dichlorofluorescein and rhodamine123, respectively. An age-dependent increase was seen
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in both cytosolic and mitochondrial ROS levels in the age groups from five to 13 months (supplementary Figs 1A and 1B). Interestingly, in one-month-old mice, the observed
4. Discussion
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ROS levels were higher than those in the five-months-old animals.
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Ageing is a complex process that affects organisms throughout their life-span, both at the cellular and the molecular level. Hence, to understand the relationship between environmental and genetic determinants that can affect ageing is a major task. DNA damage can occur from both exogenous (such as UV- or gamma-radiation or chemicals) and endogenous sources (mainly by-products of cellular metabolism, e.g., reactive oxygen species), and this DNA damage may result in cellular dysfunction and ageing [19].
In the present study, the micronucleus test and comet assay were used to study spontaneous chromosomal damage as well as DNA damage. The main objective of this work was to quantify the spontaneous occurrence of micronuclei in the bone marrow, and DNA damage in the peripheral blood in various age groups of male and female mice.
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These two methods cover different genotoxic events. The micronucleus assay detects chromosomal damage that persists for at least one mitotic cycle and is an excellent method for evaluating any damage in the genetic material. The comet assay detects DNA
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breakage and alkali-labile sites, and is a very sensitive method for measuring DNA strand-breaks at the single-cell level. Moreover, it was recently shown that the comet
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assay detected nearly 90% of the carcinogens that were negative or equivocal in the
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micronucleus assay [13]. Therefore, a combination of the micronucleus assay and the comet assay is recommended for the broad assessment of in-vivo genotoxic potential.
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The US National Toxicological Program is presently using this combined protocol as part of its efforts to evaluate the genotoxicity of substances that are a public health concern
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[20].
In our study, we have observed an age-dependent increase in the spontaneous
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micronucleus frequency in bone marrow, i.e. in Mn-PCEs and Mn-NCEs (Tables 1-3).
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There was a significantly higher incidence of micronuclei in 12- and 20-months-old mice compared with one-month-old mice. In bone marrow, the observed frequency of MnPCEs was higher than that of Mn-NCEs. This may be because the Mn-PCEs may escape directly into the peripheral blood and undergo maturation [21]. It could also be due to the selective elimination of PCEs that are harbouring micronuclei. Similar to micronucleus formation, also DNA-damage induction showed an age-
dependent increase, as was reflected in tail-moment values obtained with the comet assay (Fig. 2). Accumulation of DNA damage at older age could be due to a reduced repair capacity with age and a high susceptibility to DNA damage upon external environmental exposure [22, 23]. An age-related increase in DNA double-strand breakage has also been
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observed in human sperm [24]. Kempf et al. showed a decline in unscheduled DNA synthesis, a measure of DNA repair, in mice that were from 7 to 132 weeks old [25]. These observations also lend support to the hypothesis that the decrease in DNA repair
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may be one of the important cellular processes responsible for ageing [26]. Apart from the slowdown in DNA-repair capacity, an age-dependent increase in DNA damage may
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also be due to increased ROS accumulation upon ageing. When we measured the
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cytosolic and mitochondrial ROS levels in splenocytes of mice belonging to different age groups (1, 5, 10, 11 and 13 months), there was an age-dependent increase in ROS
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between 5 and 13 months (supplementary Figs. 1A, 1B). But in one-month-old mice the observed ROS levels were higher than in the five-months-old animals, which may be due
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to the higher metabolism and cell proliferation in younger animals. According to the free-radical theory, ageing is the cumulative result of oxidative
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damage to cells that arises due to aerobic metabolism [27]. Increased ROS accumulation
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during ageing can be attributed to the declining activity of antioxidant enzymes such as Cu/Zn-superoxide dismutase (SOD), Mn-SOD, catalase and glutathione peroxidase (Gpx) in various tissues [28-30]. Tian et al. [31] studied the level of oxidative protein damage and activities of antioxidant enzymes in different tissues such as brain, liver, heart, kidney and serum from rats at age 1, 6, 12, 18 and 24 months, and found that the level of oxidative protein damage in brain and liver was significantly higher in older rats than in younger rats. In another study, Sandhu & Kaur [32] used rats of different age groups (1, 3, 12 and 24 months) and showed that the activity level of antioxidant enzymes as well as glutathione (GSH) content in brain and lymphocytes declined with age. Likewise, Ying
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Chen et al., [33] showed gender-related differences in activities of various oxidative stress-dependent enzymes in different tissues of mice. Apart from the declining antioxidant enzyme levels, the caloric intake also is one
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of the key factors that can determine ROS accumulation in the cell. Higher calorie consumption leads to higher generation of superoxide and hydroxyl radicals, resulting in
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increased lipid peroxidation [34]. This higher level of ROS can lead to random molecular
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damage to DNA and to mitochondrial dysfunction with increasing age [35]. This damage caused to the biomolecules can be an indicator of ageing. In our study, DNA damage
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measured with the micronucleus test and the comet assay correlated well with ageing. Single-gene mutation can also be one of the indicators of the ageing process. A study by
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Ono et al., [36] has shown that the spontaneous mutation frequency of the lacZ gene in transgenic mice increased linearly with age from newborn to 12 months. But the actual
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investigated.
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gene mutations, which lead to the ageing process, are still elusive and need to be
In our study, female mice showed higher micronucleus frequencies and DNA
damage than males in all age groups. Gender is another important confounding factor that may show variation in the response to environmental mutagens. A significant gender effect on baseline micronucleus frequency favouring females has been previously reported in humans [37, 38]. On this basis, it is hypothesized that X-chromosomes play an important role in the occurrence of micronuclei and studies have also demonstrated that the inactive X-chromosome is preferentially included in the micronuclei [39]. In a study by Xuqi Chen et al., [40] female mice showed a higher food intake and accumulated more adipose tissues leading to increased body weight than male mice. This
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difference in adiposity and associated metabolic differences was proportional to the number of X chromosomes. Because of this metabolic difference, there would be higher accumulation of oxidative damage in female mice. This may be one of the reasons for the
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higher induction of micronuclei observed in females in our study.
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Conclusions
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Both the micronucleus test and the comet assay were observed to be informative, and their usefulness should be considered for the evaluation of spontaneous or induced
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genotoxicity in mice. Our results show that there is an accumulation of genetic damage in mice, as assessed by the micronucleus test and the comet assay during ageing, and that
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females show more alterations than males. Thus, ageing and gender are important
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mutagenesis or carcinogenesis.
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confounding factors that need to be comprehensively examined for evaluation of
Acknowledgements
The authors wish to thank Dr. J.R. Bandekar, Head, Radiation Biology and Health
Sciences Division for his keen interest and support. Authors also would like to thank Dr. S. Santosh Kumar, for critical reading of the manuscript. Thanks are also due to Shri. B.A. Naidu for excellent technical assistance. Authors thank the Department of Atomic Energy, India, for funding this research.
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Declaration of Interests The authors report no conflict of interests. The authors alone are responsible for the
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content and writing of the paper.
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[30] E. Xia, G. Rao, H. Van Remmen, A.R. Heydari, A. Richardson, Activities of antioxidant enzymes in various tissues of male Fischer 344 rats are altered by food restriction, J Nutr, 125 (1995) 195-201. [31] L. Tian, Q. Cai, H. Wei, Alterations of antioxidant enzymes and oxidative damage to macromolecules in different organs of rats during aging, Free radical biology & medicine, 24 (1998) 1477-1484. [32] S.K. Sandhu, G. Kaur, Alterations in oxidative stress scavenger system in aging rat brain and lymphocytes, Biogerontology, 3 (2002) 161-173. [33] Y. Chen, L.L. Ji, T.Y. Liu, Z.T. Wang, Evaluation of gender-related differences in various oxidative stress enzymes in mice, The Chinese journal of physiology, 54 (2011) 385-390. [34] D.W. Lee, B.P. Yu, Modulation of free radicals and superoxide dismutases by age and dietary restriction, Aging, 2 (1990) 357-362. [35] H.D. Osiewacz, D. Bernhardt, Mitochondrial Quality Control: Impact on Aging and Life Span - A Mini-Review, Gerontology, (2013). [36] T. Ono, Y. Miyamura, H. Ikehata, H. Yamanaka, A. Kurishita, K. Yamamoto, T. Suzuki, T. Nohmi, M. Hayashi, T. Sofuni, Spontaneous mutant frequency of lacZ gene in spleen of transgenic mouse increases with age, Mutation research, 338 (1995) 183-188. [37] M. Fenech, J. Rinaldi, The relationship between micronuclei in human lymphocytes and plasma levels of vitamin C, vitamin E, vitamin B12 and folic acid, Carcinogenesis, 15 (1994) 1405-1411. [38] J.D. Tucker, J. Nath, J.C. Hando, Activation status of the X chromosome in human micronucleated lymphocytes, Hum Genet, 97 (1996) 471-475. [39] G. Joksic, S. Petrovic, Z. Ilic, Age-related changes in radiation-induced micronuclei among healthy adults, Braz J Med Biol Res, 37 (2004) 1111-1117. [40] X. Chen, R. McClusky, J. Chen, S.W. Beaven, P. Tontonoz, A.P. Arnold, K. Reue, The number of x chromosomes causes sex differences in adiposity in mice, PLoS genetics, 8 (2012) e1002709.
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Figure legends Fig.1. Ratio of micronucleated erythrocytes between females and males observed in different age groups. Frequency of micronucleated erythrocytes in male and female mice
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were calculated by addition of micronuclei observed in polychromatic erythrocytes and
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normochromatic erythrocytes.
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Fig.2. Single-cell gel electrophoretic analysis of spontaneous DNA damage in peripheral blood leukocytes in mice of different age. DNA damage in terms of ‘tail moment’ was
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analyzed by use of SCGE-Pro software as described in the Methods section. Data are
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mice of the same age group.
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presented as the mean± S.E.M. (n=4). * p < 0.05, **p < 0.01 in comparison with male
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Fig.3. DNA damage in terms of ‘tail moment’ in male and female mice obtained at different age groups, fitted with linear regression line. Error bars indicate the S.E.M. (n=4).
Supplementary figure legend
Fig. S1. Reactive oxygen species (ROS) levels seen in cytosol (A) and mitochondria (B) using dichlorofluorescein and rhodamine123, respectively. For measuring the ROS, splenocytes were isolated from different age groups of mice, and 5x105 cells were treated with either DCFDA (10µM) or DHR123 (10µM) for 30 min before reading the fluorescence. The experiment was done in triplicate and the mean ± SEM obtained for the mice from the same group was plotted. *p < 0.05.
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Table 1. Frequency of micronucleated erythrocytes in the bone marrow of male and female Swiss mice of different age
Age (months) Sex
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_______________________________________________________________________ ‰ Mn-Es
‰ Mn-PCEs ‰ Mn-NCEs PCEs/NCEs
12
M (7)
(31/20212)
1.66±0.29
2.56±0.61
(42/25262)
(32/12484)
1.86±0.56*
2.81±0.74** 0.94±0.62
(54/29066)
(40/14211)
M (6)
0.78±0.41
(39/12129)
(58/14248)
0.96
(14/14855) 1.02
(15/14027) 1.00
(14/12106)
2.62±0.53** 4.07±0.89** 1.18±0.34 (75/28673)
0.98
(10/12778)
2.19±0.81** 3.22±0.82** 1.16±0.59
(53/24235)
F (7)
(53/14303)
1.04
(14/19359)
2.40±0.58** 3.71±0.69** 1.07±0.61 (68/28330)
20
0.72±0.57
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(45/39571)
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F (7)
1.53±0.94
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F (6)
1.14±0.51
M
M (10)
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1
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________________________________________________________________________
0.99
(17/14425)
_____________________________________________________________________ Figures within parentheses are actual numbers of micronucleated cells/total erythrocytes scored. Mn-PCEs and Mn-NCEs are average micronucleated polychromatic and micronucleated erythrocytes ± standard deviation, respectively. Mn-Es include both Mn-
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PCEs and Mn-NCEs. Values marked with asterisks (12- and 20-month-old mice) are
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significantly higher than those from the one-month-olds (*p < 0.05, **p < 0.01).
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Table 2. Statistical analysis of spontaneous frequency of micronucleated polychromatic erythrocytes in male and female mice of different age ________________________________________________________________________ Sex
‰ Mn-PCEs Variance
N
t-values
p-values Significance
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Age
at (p = 0.05)
0.870
10
12
M
2.81
0.541
7
1
M
1.53
0.870
10
20
M
3.22
0.670
6
12
M
2.81
0.541
7
20
M
3.22
0.670
1
F
2.56
0.369
6
12
F
3.71
0.479
7
F
2.56
0.369
6
F
4.07
0.794
7
F
3.71
0.479
7
F
4.07
0.794
7
20 12 20
6
d
Ac ce pt e
1
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1.53
3.24
0.006
S
0.002
S
0.93
0.372
NS
3.05
0.011
S
3.49
0.005
S
0.92
0.375
NS
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M
3.81
M
1
cr
________________________________________________________________________
________________________________________________________________________ S: Significantly different; NS: Not significantly different
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Table 3.
Comparative spontaneous frequency of micronucleated polychromatic
erythrocytes between male and female mice of different age _______________________________________________________________________ Sex
‰ Mn-PCEs Variance
N
t-values
p-values Significance
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Age
at (p = 0.05)
0.870
10
1
F
2.56
0.369
6
12
M
2.81
0.541
7
12
F
3.71
0.479
7
20
M
3.22
0.670
6
20
F
4.07
0.794
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1.53
2.40
an
M
2.27
M
1
cr
________________________________________________________________________
7
1.79
0.031
S
0.043
S
0.100
NS
d
________________________________________________________________________
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S: Significantly different; NS: Not significantly different
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Table 4. Slopes of the mean spontaneous frequency of micronucleated polychromatic erythrocytes in male and female mice of different age
Age (months)
Sex
Slopes/month of Mn-PCEs
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________________________________________________________________________
Males
0.116
12-20
Males
0.051
1-12
Females
0.105
12-20
Females
an
us
1-12
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________________________________________________________________________
0.045
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d
M
________________________________________________________________________
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S1383-5718(14)00160-0 http://dx.doi.org/doi:10.1016/j.mrgentox.2014.04.026 MUTGEN 402497
To appear in:
Mutation Research
Received date: Revised date: Accepted date:
20-8-2013 25-3-2014 30-4-2014
Please cite this article as: H.N. Bhilwade, S. Jayakumar, R.C. Chaubey, Agedependent changes in spontaneous frequency of micronucleated erythrocytes in bone marrow and DNA damage in peripheral blood of Swiss mice, Mutation Research/Genetic Toxicology and Environmental Mutagenesis (2014), http://dx.doi.org/10.1016/j.mrgentox.2014.04.026 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Highlights: 1. Ageing enhanced the bone‐marrow micronucleated erythrocytes in mice 2. As compared to males, females showed higher micronuclei frequency with age
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3. Enhancement of DNA damage was also observed with age in mice
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Age-dependent changes in spontaneous frequency of micronucleated
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Swiss mice
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erythrocytes in bone marrow and DNA damage in peripheral blood of
*
Hari N.Bhilwade , S. Jayakumar, R.C.Chaubey Radiation Biology and Health Sciences Division, Bhabha Atomic Research Centre,
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Mumbai 400 085, India
*Corresponding author
Radiation Biology and Health Sciences Division Bhabha Atomic Research Centre Mumbai 400 085, India
Email: [email protected] Tel.: +91-22-2559-0425 FAX: +91-22- 2550-5151
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Abstract
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Age-dependent changes in chromosomal damage in bone marrow – a selfproliferating tissue – in the form of spontaneously occurring micronucleated erythrocytes,
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and DNA damage in peripheral blood were examined in male and female Swiss mice. In the erythrocyte population in the bone marrow, polychromatic (immature) erythrocytes
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showed a significant increase in the frequency of micronuclei as a function of age of the mice (1–20 months). The increase in micronucleus frequency was less in normochromatic
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(mature) erythrocytes. The female mice showed a higher frequency of micronuclei than
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the male mice in all the age groups examined. However, the female to male ratio of micronucleus frequencies in total erythrocytes as well as in polychromatic erythrocytes decreased with age. DNA damage, measured as tail moment in the single-cell gel electrophoresis in peripheral blood of different age groups of mice (1, 6, 12 and 18 months) showed a gradual increase with age. Female mice showed more DNA damage than 1-month and 18-months old male mice. In conclusion, these results show that there is an accumulation of genetic damage in bone marrow and DNA damage in peripheral blood of mice during ageing, and that females show more alterations than males.
Keywords: Ageing, DNA damage, micronucleus assay, comet assay
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1. Introduction Age is one of the important confounding factors that influence the response of an organism to physical, chemical and other environmental agents. Ageing is a natural
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process in which the functions of tissues and organs of the body diminish with advancing age. The exact mechanism of ageing is unclear, but the hypothesis that ‘ageing is a
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consequence of alterations of cellular macromolecules’ is supported by several studies at
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the cellular and tissue level of higher organisms [1]. DNA, the genetic material, is the primary object of studies related to the ageing process. The somatic mutation theory of
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ageing tries to explain the process as a function of genetic alterations in somatic cells, mainly involving DNA and deterioration of chromatin organization [1, 2]. In a number of
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studies, changes in chromosomal DNA are correlated with ageing, both in human and animal models [3], by use of various cytogenetic parameters such as chromosomal
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aberrations, sister chromatid exchange and micronuclei [4]. Using the micronucleus
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assay, Ganguly et al., demonstrated that the frequency of micronuclei in bi-nucleated cells of human peripheral lymphocytes increased with age [5]. On the contrary Fenech et al., observed no such age-dependent increase in micronucleus frequency in humans [6]. On the other hand, Orta & Gunebaken observed an increase in micronucleus frequency until the age of 50 and then a decrease up to the age of 73 in humans [7]. Besides these human studies, several investigations have been carried out in mouse models regarding changes in spontaneous frequency of micronuclei upon ageing [8-12]. Among these, some have shown an age-dependent increase in the micronucleus frequency in mice [8, 11]. However, in other studies with mice and rats, such an increase could not be observed [9, 10, 12]. Similar inconsistencies have also been reported with other cytogenetic
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endpoints in human and animal models, suggesting the need for further studies in this area, with a combination of different techniques. This will help build consensus on the correlation between ageing and different cytogenetic parameters. Besides, the
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information on changes in baseline frequency of cytogenetic endpoints with advancing age can be useful when comparing situations when baseline frequencies of exposed
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individuals are not known prior to accidental exposure [4]. Moreover, it would also help
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in comparing environmental risk or exposure in different age groups.
In the present study, we have combined the micronucleus test and the comet assay
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to study the spontaneous micronucleus frequency in bone marrow and the baseline level of DNA damage in peripheral blood, respectively, in different age groups of mice. The
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frequency of micronuclei is a reliable measure of both chromosome loss and breakage, but can only be conducted in dividing cells. On the other hand, the comet assay can detect
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a broad spectrum of DNA damage (including DNA single- and double-strand breaks,
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base damage, alkali-labile DNA adducts, and other DNA lesions associated with diverse reactive oxygen species) virtually in any tissue [13]. Since there is paucity of data on changes in incidence of micronuclei in the bone
marrow as well as DNA strand-breaks in mice as a function of age, this study was aimed to examine the frequency of spontaneously occurring micronucleated erythrocytes in bone marrow by means of the micronucleus test, and of DNA strand-breaks with the comet assay, in different age groups of mice. The role of sex on the incidence of chromosomal/DNA damage with advancing age has also been analyzed.
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2. Materials and methods 2.1 Materials Fetal calf serum (GIBCO, USA), May-Gruenwald and Giemsa stains, SYBR-
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green-II stain, low-melting agarose, ethylenediammine tetraacetic acid (Na2-EDTA), Triton X-100, dimethyl sulfoxide (DMSO), Tris HCl, (Sigma Chemicals, USA),
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methanol, xylene (Qualigens fine chemicals, Glaxo, Bombay, India), NaCl (SD Fine
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chemicals, India), NaOH (Thomas Baker Chemicals Ltd, India) and fully frosted slides (Erie Scientific, Sybron, Portsmouth, N.H. USA) were used in these studies.
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2.2 Animals
Inbred albino Swiss male and female mice reared in the animal house of Bhabha
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Atomic Research Centre, Mumbai were used in this study. All the animals were caged individually and maintained under controlled conditions of temperature (23-25°C), and a
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libitum.
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12h/12h light/dark cycle. Animals received nutritionally balanced food and water ad
2.3 Micronucleus test
Six to ten mice of either sex of different age groups (1, 12 and 20 months) were
used for the bone-marrow micronucleus assay. Animals were processed for enumerating micronuclei in the erythrocytes as described previously [14] with minor modifications as reported earlier [15]. In brief, bone marrow was flushed in a 5-ml centrifuge tube containing fetal bovine serum and centrifuged at 1500 rpm [give proper ‘x g’-value here; Ed.] for 5 min. The cell pellet was mixed thoroughly, bone-marrow smears were made on clean glass slides and stained as follows: 5-min incubation in undiluted MayGruenwald (0.25% in methanol), 3 min in diluted May–Gruenwald solution (1:1, May–
Page 6 of 24
Gruenwald/distilled water), rinsed in distilled water three times and then stained with diluted Giemsa (1:6 of the Giemsa stock/distilled water) for 10 min following [followed by? Ed.] thorough washing with distilled water. The slides were dried, cleared for 5 min
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in xylene, and mounted in DPX. Two slides were made per animal. Coded slides were scored for the incidence of micronucleated polychromatic erythrocytes (Mn-PCEs) and
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micronucleated normochromatic erythrocytes (Mn-NCEs) at 100x magnification under
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oil-immersion. At least 2000 polychromatic erythrocytes (PCEs) with or without micronuclei and a corresponding number of normochromatic erythrocytes (NCEs) were
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scored per animal. 2.4 Alkaline comet assay
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For detection of total DNA strand-breaks in peripheral blood leukocytes, the alkaline comet assay (single-cell gel electrophoresis) was used [16]. For this assay, about
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50 µl of heparinised whole blood from four mice (two males, two females) belonging to
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different age groups (1, 6, 12 and 18 months) were collected and mixed with 1.0 ml of 0.8% low-melting agarose solution prepared in 0.9% saline at 38ºC, and evenly layered on fully-frosted slides. After solidification of the agarose, the slides were kept in lysis buffer (2.5 M NaCl, 100 mM Na2-EDTA, 1% Triton X-100, 10 mM Tris-HCl [pH? Ed.] and 10% DMSO) for 1h at 4ºC. The slides were removed from the lysis solution, washed with alkaline buffer, and placed on a horizontal electrophoresis apparatus in alkaline buffer (300 mM NaOH, 1 mM Na2-EDTA, pH 13.0) at room temperature for 20 min to allow DNA unwinding and expression of alkali-labile sites. Electrophoresis was carried out for 20 minutes at 0.8V/cm. Afterwards, the slides were washed gently in neutralizing buffer (0.4 M Tris-HCl, pH 7.5) to remove alkali and detergents, stained with SYBR
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Green II dye and observed under a fluorescence microscope (Carl Zeiss Axio-vision) at 40x magnification. Images of 50-60 individual cells per slide were acquired with a digital imaging system and stored as bitmap file. These images were analyzed with SCGE-PRO
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software [17] and different parameters of DNA damage such as % DNA in tail (%DNAT), tail length (TL) and tail moment (TM, product of fraction of DNA in tail and tail
2.5. Measurement of Reactive Oxygen Species levels
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have used tail moment for representing DNA damage [18].
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length) were obtained. As the tail moment is considered as the optimum parameter, we
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Reactive oxygen species (ROS) levels in cytosol and mitochondria were measured with dichlorofluorescein and rhodamine123, respectively. For measurement of ROS,
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splenocytes were isolated from male Balb/C mice of different age: 1 month (N=4), 5 months (N=4), 10 months (N=2), 11 months (N=2), and 13 months (N=2), and 5x10
5
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cells were placed in 96-well plates and treated with either dichloro-dihydrofluorescein
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diacetate (DCFDA - 10µM) or dihydrorhodamine123 (DHR123 - 10µM). Thirty min after the treatment, fluorescence was read with a fluorimeter (DCF: λex 485nm and λem 535nm; Rhodamine123: λex 511nm and λem 535nm). 2.6. Statistical analysis
In the micronucleus test and the comet assay, means and standard deviations were
calculated based on the micronucleus frequency or the tail-moment values obtained from individual animals of the particular age group. Statistical significance between the groups was analysed by use of the t-test and analysis of variance (ANOVA), with GraphPad Prism 5.0. Values were considered significant at p < 0.05.
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3. Results 3.1. Micronucleus frequency increases with advancing age in mice The micronucleus assay was conducted in 1-, 12- and 20-month old Swiss mice,
dependent
increase
in
micronucleus
frequency
both
in
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to determine whether the incidence of micronuclei varied with age. We observed an agepolychromatic
and
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normochromatic erythrocytes of the bone marrow. The frequencies of Mn-PCEs, Mn-
NCEs, and total micronucleated erythrocytes (Mn-Es) in the bone marrow of male and
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female mice in different age groups are summarized in Table 1.
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In male mice, a significant increase in the frequency of Mn-PCEs per thousand cells was observed at 12 months (2.81±0.74) and 20 months (3.22±0.82) compared with
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that of one-month-old mice (1.53 ±0.94). Similar to males, female mice also showed a significant increase in spontaneous frequency of Mn-PCEs upon ageing. The spontaneous
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frequency of Mn-PCEs in one-month-old female mice was 2.56±0.61, compared with
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3.71±0.69 at 12 months and 4.07±0.89 at 20 months (Table 1, Table 2). The frequency of Mn-NCEs and Mn-Es followed the same trend as Mn-PCEs, both in male and female mice. However, the frequency of micronuclei observed in NCEs was lower than that in PCEs in all age groups, both in male and female mice. When we compared the difference in micronucleus frequency between 12- and 20-months-old mice, Mn-PCEs, Mn-NCEs and Mn-Es did not change significantly. The PCEs/NCEs ratio, reflecting the proliferative activity of the bone-marrow cells, remained comparable between males and females of different age groups. Another interesting observation in this study was that the female mice showed a higher frequency of Mn-PCEs, Mn-NCEs and Mn-Es than the males, in all age groups (Table 1, Table 3). The frequency of Mn-PCEs in one-month-old female mice was
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2.56±0.61, which was significantly higher than the corresponding frequency of Mn-PCEs in one-month-old male mice (1.53±0.94). Similarly, at 12 months female mice showed a significantly higher frequency of Mn-PCEs than the male mice. However, at 20 months,
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although the female mice still showed a higher Mn-PCE frequency than the males, it was no longer significantly different. The ratio of Mn-Es frequency between female and male
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the frequencies at 12 months (1.29) and 20 months (1.14).
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was also analyzed (Fig.1) and at one month it was found to be highest (1.46), followed by
The slope depicting the mean spontaneous frequency of Mn-PCEs in male and
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females with increasing age was also calculated (Table 4). The rate of induction of MnPCEs was relatively high in the younger age groups (1-12 months) in both males and
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females compared with the older age groups (12-20 months) of either sex, which was reflected by a steeper slope. The slope for Mn-PCEs in males was higher than that of
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females in younger (1-12 months) as well as older (12-20 months) age groups.
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3.2. DNA damage increases with advancing age in mice DNA damage in terms of tail moment observed in male and female mice of
different age viz. 1, 6, 12 and 18 months, was measured by use of the alkaline comet assay (Fig. 2). Similar to the micronucleus frequency in the bone marrow, the spontaneous DNA damage also showed a statistically significant, age-dependent increase in male mice. The tail moment observed in one-month-old male mice was 1.33±0.16, which had increased more than two-fold at six months (3.17±0.12), and it continued to increase up to 18 months. But the rate of increase in tail moment was comparatively slow between 12 and 18 months. Likewise, in females a similar age-dependent increase in spontaneous DNA damage was observed, as reflected in tail-moment values in the comet
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assay. The statistical parameters characterizing the DNA-damage response (tail moment) with age in male and female mice were calculated (Fig.3). In the age-dependent increase in DNA damage, the males showed a steeper slope (0.153) than the females (0.121).
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3.3. Levels of reactive oxygen species (ROS) show age-dependent increase in mice ROS levels are a good measure of oxidative stress in the cell. We have measured
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the cytosolic and mitochondrial ROS levels in splenocytes of mice belonging to different
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age groups (1, 5, 10, 11 and 13 months) by measuring the fluorescence of dichlorofluorescein and rhodamine123, respectively. An age-dependent increase was seen
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in both cytosolic and mitochondrial ROS levels in the age groups from five to 13 months (supplementary Figs 1A and 1B). Interestingly, in one-month-old mice, the observed
4. Discussion
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ROS levels were higher than those in the five-months-old animals.
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Ageing is a complex process that affects organisms throughout their life-span, both at the cellular and the molecular level. Hence, to understand the relationship between environmental and genetic determinants that can affect ageing is a major task. DNA damage can occur from both exogenous (such as UV- or gamma-radiation or chemicals) and endogenous sources (mainly by-products of cellular metabolism, e.g., reactive oxygen species), and this DNA damage may result in cellular dysfunction and ageing [19].
In the present study, the micronucleus test and comet assay were used to study spontaneous chromosomal damage as well as DNA damage. The main objective of this work was to quantify the spontaneous occurrence of micronuclei in the bone marrow, and DNA damage in the peripheral blood in various age groups of male and female mice.
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These two methods cover different genotoxic events. The micronucleus assay detects chromosomal damage that persists for at least one mitotic cycle and is an excellent method for evaluating any damage in the genetic material. The comet assay detects DNA
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breakage and alkali-labile sites, and is a very sensitive method for measuring DNA strand-breaks at the single-cell level. Moreover, it was recently shown that the comet
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assay detected nearly 90% of the carcinogens that were negative or equivocal in the
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micronucleus assay [13]. Therefore, a combination of the micronucleus assay and the comet assay is recommended for the broad assessment of in-vivo genotoxic potential.
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The US National Toxicological Program is presently using this combined protocol as part of its efforts to evaluate the genotoxicity of substances that are a public health concern
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[20].
In our study, we have observed an age-dependent increase in the spontaneous
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micronucleus frequency in bone marrow, i.e. in Mn-PCEs and Mn-NCEs (Tables 1-3).
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There was a significantly higher incidence of micronuclei in 12- and 20-months-old mice compared with one-month-old mice. In bone marrow, the observed frequency of MnPCEs was higher than that of Mn-NCEs. This may be because the Mn-PCEs may escape directly into the peripheral blood and undergo maturation [21]. It could also be due to the selective elimination of PCEs that are harbouring micronuclei. Similar to micronucleus formation, also DNA-damage induction showed an age-
dependent increase, as was reflected in tail-moment values obtained with the comet assay (Fig. 2). Accumulation of DNA damage at older age could be due to a reduced repair capacity with age and a high susceptibility to DNA damage upon external environmental exposure [22, 23]. An age-related increase in DNA double-strand breakage has also been
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observed in human sperm [24]. Kempf et al. showed a decline in unscheduled DNA synthesis, a measure of DNA repair, in mice that were from 7 to 132 weeks old [25]. These observations also lend support to the hypothesis that the decrease in DNA repair
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may be one of the important cellular processes responsible for ageing [26]. Apart from the slowdown in DNA-repair capacity, an age-dependent increase in DNA damage may
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also be due to increased ROS accumulation upon ageing. When we measured the
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cytosolic and mitochondrial ROS levels in splenocytes of mice belonging to different age groups (1, 5, 10, 11 and 13 months), there was an age-dependent increase in ROS
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between 5 and 13 months (supplementary Figs. 1A, 1B). But in one-month-old mice the observed ROS levels were higher than in the five-months-old animals, which may be due
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to the higher metabolism and cell proliferation in younger animals. According to the free-radical theory, ageing is the cumulative result of oxidative
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damage to cells that arises due to aerobic metabolism [27]. Increased ROS accumulation
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during ageing can be attributed to the declining activity of antioxidant enzymes such as Cu/Zn-superoxide dismutase (SOD), Mn-SOD, catalase and glutathione peroxidase (Gpx) in various tissues [28-30]. Tian et al. [31] studied the level of oxidative protein damage and activities of antioxidant enzymes in different tissues such as brain, liver, heart, kidney and serum from rats at age 1, 6, 12, 18 and 24 months, and found that the level of oxidative protein damage in brain and liver was significantly higher in older rats than in younger rats. In another study, Sandhu & Kaur [32] used rats of different age groups (1, 3, 12 and 24 months) and showed that the activity level of antioxidant enzymes as well as glutathione (GSH) content in brain and lymphocytes declined with age. Likewise, Ying
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Chen et al., [33] showed gender-related differences in activities of various oxidative stress-dependent enzymes in different tissues of mice. Apart from the declining antioxidant enzyme levels, the caloric intake also is one
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of the key factors that can determine ROS accumulation in the cell. Higher calorie consumption leads to higher generation of superoxide and hydroxyl radicals, resulting in
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increased lipid peroxidation [34]. This higher level of ROS can lead to random molecular
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damage to DNA and to mitochondrial dysfunction with increasing age [35]. This damage caused to the biomolecules can be an indicator of ageing. In our study, DNA damage
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measured with the micronucleus test and the comet assay correlated well with ageing. Single-gene mutation can also be one of the indicators of the ageing process. A study by
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Ono et al., [36] has shown that the spontaneous mutation frequency of the lacZ gene in transgenic mice increased linearly with age from newborn to 12 months. But the actual
Ac ce pt e
investigated.
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gene mutations, which lead to the ageing process, are still elusive and need to be
In our study, female mice showed higher micronucleus frequencies and DNA
damage than males in all age groups. Gender is another important confounding factor that may show variation in the response to environmental mutagens. A significant gender effect on baseline micronucleus frequency favouring females has been previously reported in humans [37, 38]. On this basis, it is hypothesized that X-chromosomes play an important role in the occurrence of micronuclei and studies have also demonstrated that the inactive X-chromosome is preferentially included in the micronuclei [39]. In a study by Xuqi Chen et al., [40] female mice showed a higher food intake and accumulated more adipose tissues leading to increased body weight than male mice. This
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difference in adiposity and associated metabolic differences was proportional to the number of X chromosomes. Because of this metabolic difference, there would be higher accumulation of oxidative damage in female mice. This may be one of the reasons for the
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higher induction of micronuclei observed in females in our study.
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Conclusions
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Both the micronucleus test and the comet assay were observed to be informative, and their usefulness should be considered for the evaluation of spontaneous or induced
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genotoxicity in mice. Our results show that there is an accumulation of genetic damage in mice, as assessed by the micronucleus test and the comet assay during ageing, and that
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females show more alterations than males. Thus, ageing and gender are important
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mutagenesis or carcinogenesis.
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confounding factors that need to be comprehensively examined for evaluation of
Acknowledgements
The authors wish to thank Dr. J.R. Bandekar, Head, Radiation Biology and Health
Sciences Division for his keen interest and support. Authors also would like to thank Dr. S. Santosh Kumar, for critical reading of the manuscript. Thanks are also due to Shri. B.A. Naidu for excellent technical assistance. Authors thank the Department of Atomic Energy, India, for funding this research.
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Declaration of Interests The authors report no conflict of interests. The authors alone are responsible for the
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content and writing of the paper.
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Figure legends Fig.1. Ratio of micronucleated erythrocytes between females and males observed in different age groups. Frequency of micronucleated erythrocytes in male and female mice
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were calculated by addition of micronuclei observed in polychromatic erythrocytes and
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normochromatic erythrocytes.
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Fig.2. Single-cell gel electrophoretic analysis of spontaneous DNA damage in peripheral blood leukocytes in mice of different age. DNA damage in terms of ‘tail moment’ was
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analyzed by use of SCGE-Pro software as described in the Methods section. Data are
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mice of the same age group.
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presented as the mean± S.E.M. (n=4). * p < 0.05, **p < 0.01 in comparison with male
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Fig.3. DNA damage in terms of ‘tail moment’ in male and female mice obtained at different age groups, fitted with linear regression line. Error bars indicate the S.E.M. (n=4).
Supplementary figure legend
Fig. S1. Reactive oxygen species (ROS) levels seen in cytosol (A) and mitochondria (B) using dichlorofluorescein and rhodamine123, respectively. For measuring the ROS, splenocytes were isolated from different age groups of mice, and 5x105 cells were treated with either DCFDA (10µM) or DHR123 (10µM) for 30 min before reading the fluorescence. The experiment was done in triplicate and the mean ± SEM obtained for the mice from the same group was plotted. *p < 0.05.
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Table 1. Frequency of micronucleated erythrocytes in the bone marrow of male and female Swiss mice of different age
Age (months) Sex
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_______________________________________________________________________ ‰ Mn-Es
‰ Mn-PCEs ‰ Mn-NCEs PCEs/NCEs
12
M (7)
(31/20212)
1.66±0.29
2.56±0.61
(42/25262)
(32/12484)
1.86±0.56*
2.81±0.74** 0.94±0.62
(54/29066)
(40/14211)
M (6)
0.78±0.41
(39/12129)
(58/14248)
0.96
(14/14855) 1.02
(15/14027) 1.00
(14/12106)
2.62±0.53** 4.07±0.89** 1.18±0.34 (75/28673)
0.98
(10/12778)
2.19±0.81** 3.22±0.82** 1.16±0.59
(53/24235)
F (7)
(53/14303)
1.04
(14/19359)
2.40±0.58** 3.71±0.69** 1.07±0.61 (68/28330)
20
0.72±0.57
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(45/39571)
Ac ce pt e
F (7)
1.53±0.94
an
F (6)
1.14±0.51
M
M (10)
d
1
cr
________________________________________________________________________
0.99
(17/14425)
_____________________________________________________________________ Figures within parentheses are actual numbers of micronucleated cells/total erythrocytes scored. Mn-PCEs and Mn-NCEs are average micronucleated polychromatic and micronucleated erythrocytes ± standard deviation, respectively. Mn-Es include both Mn-
Page 20 of 24
PCEs and Mn-NCEs. Values marked with asterisks (12- and 20-month-old mice) are
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M
an
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significantly higher than those from the one-month-olds (*p < 0.05, **p < 0.01).
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Table 2. Statistical analysis of spontaneous frequency of micronucleated polychromatic erythrocytes in male and female mice of different age ________________________________________________________________________ Sex
‰ Mn-PCEs Variance
N
t-values
p-values Significance
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Age
at (p = 0.05)
0.870
10
12
M
2.81
0.541
7
1
M
1.53
0.870
10
20
M
3.22
0.670
6
12
M
2.81
0.541
7
20
M
3.22
0.670
1
F
2.56
0.369
6
12
F
3.71
0.479
7
F
2.56
0.369
6
F
4.07
0.794
7
F
3.71
0.479
7
F
4.07
0.794
7
20 12 20
6
d
Ac ce pt e
1
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1.53
3.24
0.006
S
0.002
S
0.93
0.372
NS
3.05
0.011
S
3.49
0.005
S
0.92
0.375
NS
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M
3.81
M
1
cr
________________________________________________________________________
________________________________________________________________________ S: Significantly different; NS: Not significantly different
Page 22 of 24
Table 3.
Comparative spontaneous frequency of micronucleated polychromatic
erythrocytes between male and female mice of different age _______________________________________________________________________ Sex
‰ Mn-PCEs Variance
N
t-values
p-values Significance
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Age
at (p = 0.05)
0.870
10
1
F
2.56
0.369
6
12
M
2.81
0.541
7
12
F
3.71
0.479
7
20
M
3.22
0.670
6
20
F
4.07
0.794
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1.53
2.40
an
M
2.27
M
1
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________________________________________________________________________
7
1.79
0.031
S
0.043
S
0.100
NS
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________________________________________________________________________
Ac ce pt e
S: Significantly different; NS: Not significantly different
Page 23 of 24
Table 4. Slopes of the mean spontaneous frequency of micronucleated polychromatic erythrocytes in male and female mice of different age
Age (months)
Sex
Slopes/month of Mn-PCEs
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________________________________________________________________________
Males
0.116
12-20
Males
0.051
1-12
Females
0.105
12-20
Females
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us
1-12
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________________________________________________________________________
0.045
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M
________________________________________________________________________
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