Mutagenesis Advance Access originally published online on June 4, 2007
Mutagenesis 2007 22(5):311-315; doi:10.1093/mutage/gem018
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Suitability of cryopreserved isolated lymphocytes for the analysis of micronuclei with the cytokinesis-block method
Department of Environment and Primary Prevention, Istituto Superiore di Sanità, Rome Italy
In order to assess the applicability of the cytokinesis-block micronucleus assay to frozen cells in human biomonitoring and in vitro radiosensitivity studies, basal and radiation-induced micronuclei and nucleoplasmic bridges (NPBs) were analysed in 28 lymphocyte samples stored frozen from 18 to 123 weeks. All samples successfully proliferated and produced a sufficient number of binucleated cells to be analysed. The length of the cryopreservation period did not influence cell proliferation, nor the incidence of micronuclei and NPBs, both in untreated and irradiated cells. Spontaneous levels of micronuclei were modulated by age (P = 0.007) and by gender (P = 0.024), as previously shown for cultures set up using fresh cell samples. Irradiation with 2 Gy 
-rays significantly increased both micronuclei and NPBs, which were significantly correlated to each other (P = 0.004). Radiation-induced micronuclei significantly increased with the age of donors (P = 0.035), confirming previous findings obtained with fresh cell samples. The spontaneous incidences of micronuclei observed in cultures set up with frozen lymphocytes were compared with data recorded from the same subjects 5 years before using fresh blood samples. A high correlation was observed between the two data sets (P = 0.004 after removing age and gender effects), highlighting the stability during the time of micronuclei as a biomarker of genomic stability, and supporting the suitability of frozen cells for cytogenetic analyses in biomonitoring and susceptibility studies.
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Introduction |
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The analysis of cytogenetic biomarkers of DNA damage in peripheral lymphocytes has widespread application in the biomonitoring of mutagen-exposed populations and in susceptibility studies to discriminate at-risk individuals (1
–7). To this aim, mitogen-stimulated lymphocyte cultures prepared from fresh blood samples are commonly used. In principle, the use of fresh blood provides significant practical advantages, such as the requirement of small amounts of blood (<1 ml for each lymphocyte culture) and good proliferation of cells following mitogen stimulation. However, several drawbacks limit the usefulness of fresh blood in human cytogenetic surveys. In large biomonitoring studies, for example, processing hundreds of samples in a short time can be logistically difficult; on the other hand, to arrange the collection of blood specimens over a long-time interval may results in seasonal variation (8–10) and increased experimental variability. Similarly, in investigations on biomarker prevalence and genetic polymorphisms, blood specimens are usually taken once and immediately used to set up cell cultures for cytogenetic analyses, before genotyping of study subjects. This may determine an unbalanced distribution of genotypes, and hence requires the recruitment and analysis of relatively large populations in order to guarantee a sufficient number of cases with rare alleles, adding time and costs to the conduct of the study.
These drawbacks can potentially be overcome using cryopreserved cells. In principle, the use of cryopreserved cells may allow better planning of the study, with the a priori selection of subjects with relevant genotypes, the confirmation of results in replicated experiments and the recovery of data in case of lost sample. Moreover, the use of cryopreserved cells may allow advantage to be taken of the current large diffusion of biobanks (11) to perform comparative cross-sectional studies on well-characterized subjects, or retrospective investigations on predictivity of cytogenetic biomarkers for cancer risk using biological specimens obtained before the first clinical sign of disease.
Cryopreserved, unstimulated whole blood or isolated lymphocytes have been used previously in investigations on endogenous DNA damage and/or DNA repair capability by means of the host reactivation (12,13) and comet assays (14–17). On the other hand, limited experience is available on the suitability of cryopreserved isolated lymphocytes (18,19) or whole blood (20) for the analysis of cytogenetic biomarkers. In this respect, no comparative evaluation of frozen and fresh cells in terms of background incidence of cytogenetic damages and response to in vitro challenge has so far been performed. On the other hand, elucidating whether freezing and storage affects chromosome stability of lymphocytes is mandatory if frozen cells are to be used in human cytogenetic surveys.
In this work, the effect of freezing on the performance of the cytokinesis-block micronucleus (CBMN) assay in human lymphocytes has been addressed. Frequencies of baseline and radiation-induced micronuclei were measured in cultures set up with cell samples stored frozen for 18–123 weeks, and the effects of length of storage, age, gender and other known or possible confounding factors were evaluated. Furthermore, the results with frozen cells were compared with those obtained 5 years before with fresh blood in the same subjects, in order to assess the stability over time of baseline incidence of micronuclei, as an individual trait of genomic stability.
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Materials and methods |
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Study subjects and samplings
Blood samples were provided by 28 healthy subjects. Demographic characteristics of the study subjects are summarized in Table I. All subjects gave an informed written consent for participation in the study. All analyses were carried out on anonymous, coded samples. Each subject provided a single blood specimen (10–14 ml) taken by venipuncture in heparinized vacutainers (BD, Plymouth, UK).
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Isolation and storing of lymphocytes
Whole blood was diluted in an equal volume of RPMI culture medium (Gibco, Auckland, NZ) and stratified on Histopaque-1077 (Sigma, St Louis, MO) for lymphocyte separation according to the manufacturer's instructions. After isolation, lymphocytes were washed in RPMI, diluted in 1-ml culture medium and counted in a KOVA glasstic slide 10 (Hycor, Garden Grove, CA) after trypan blue (Euroclone, Pero, IT) exclusion staining. At least 107 lymphocytes were obtained from each blood sample. Lymphocyte freezing and thawing were carried out following the procedures recommended by Risom and Knudsen (21
). Briefly, lymphocytes (1 x 107 cells/ml) were suspended in cold freezing medium containing 40% RPMI, 50% fetal bovine serum (FBS) (Euroclone) and 10% dimethyl sulfoxide (Carlo Erba, Rodano, IT), dispensed in at least three cryogenic vials (350–800 µl per vial, depending on cell availability), stored at –80°C for 24 h and then transferred in liquid nitrogen until use.
Cell cultures
To set up cell lymphocytes cultures for cytogenetic analyses, one aliquot of cryopreserved cell suspension was thawed quickly in a water bath at 37°C and suspended in 5 ml cold medium containing 50% FBS, 49% RPMI and 1% of dextrose. Cells were spun down by centrifugation (10 min at 1000 r.p.m.) at 4°C, counted in a glasstic slide and suspended at 0.5 x 106 to 1.0 x 106 viable cells/ml in 0.75 ml RPMI containing 15% FBS and 2% phytohaemoagglutinin (PHA) (Murex, Dartford, UK). For each blood sample at least six cultures were set up in round-bottomed tissue cultures tubes. Two to four lymphocyte samples were analysed in one experiment.
Treatment with ionizing radiation
Three cell cultures for each subject were irradiated with -rays before the beginning of incubation at 37°C. Treatment conditions were as follows: 2 Gy from a 137Cs source at the dose rate of 2 Gy/min.
CBMN test
Irradiated and untreated lymphocyte cultures were incubated at 37°C with loose lids in a humidified atmosphere containing 5% CO2. Forty-four hours after PHA stimulation, cytochalasin B (Cyt B) (Sigma) was added at the final concentration of 4.5 µg/ml. Cells were collected onto slides by by Shandon Cytospin® 3 (Thermo Electron Corporation, Waltham, MA) 72 h after stimulation, then fixed for 10 min in absolute methanol and stained in 5% Giemsa (Carlo Erba) solution. For each subject, 1000 binucleated cells were scored from both irradiated and untreated cultures (500 cells from each of two cultures). Scoring of binucleated cells with micronuclei (MnBin) and nucleoplasmic bridge (NPB) was performed following the criteria developed by Fenech et al. (22) in the framework of the HUMN project. Cell proliferation was evaluated determining the nuclear division index (NDI) in 200 cells. NDI was calculated as follows: (number of binucleated cells + twice the number of multinucleated cells) / total number of scored cells.
Statistical analysis
The influence of possible explicative variables on the incidences of micronuclei and NPB and on the NDI of cell cultures was evaluated by multiple and stepwise regression analysis. In order to stabilize variance, data deviating from normality were log transformed. All analyses were carried out with the SPSS statistical package (version 13.0).
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Results |
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Baseline incidence of cytogenetic biomarkers
The results of CBMN assays with untreated cell cultures set up with 28 frozen lymphocyte samples are summarized in Table II. All cultures successfully proliferated and produced a sufficient number of binucleated cells to allow the scoring of 1000 binucleated cells. The average NDI value of cultures (0.64) indicated that >50% of stimulated lymphocytes divided successfully after Cyt B addition. The overall incidence of micronucleated cells (MnBin) was close to values reported in the literature and observed in previous investigations in this laboratory with fresh whole blood and isolated lymphocytes (23
,24). Due to a couple of outliers, the distribution of individual values deviated from normality and data were natural ln transformed for statistical analysis. NPBs were rarely observed, as conceivable for a marker of chromosome-type aberrations (dicentric and centric ring) (25).
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The analysis of data by stepwise multiple regression analysis showed that both gender and age significantly modulated the frequency of MnBin, while no significant influence of the storing period or proliferation rate, expressed as NDI, was disclosed (Table III). In line with previous observations with fresh blood samples, also in cultures set up with frozen isolated lymphocytes the incidence of MnBin was higher in female subjects and correlated positively with the age of donor at the time of sampling (Figure 1). On the other hand, none of the parameters considered proved to affect NPB frequency, which was also not correlated with MnBin frequency. However, statistical analysis of NPB data was flawed by the low number of events recorded due to the very low spontaneous frequency of NPB in cytokinesis-blocked human lymphocytes. Finally, none of the explicative variables considered, including the time of storage under liquid nitrogen, were shown to affect cell proliferation as evaluated by NDI.
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Incidence of cytogenetic biomarkers in irradiated cultures
Data from irradiated lymphocyte cultures are summarized in Table IV. After treatment with 2 Gy
-rays, both MnBin and NPB were significantly increased in all cultures (P < 0.001) and significantly correlated with each other (P = 0.004). To a lesser extent, but still highly significant (P < 0.001), NDI was also affected by the treatment. Multiple regression analysis did not highlight any significant effects of the length of cryopreservation period, NDI, gender, age and spontaneous values on the incidence of MnBin and NPB in irradiated cultures (data not shown); however, stepwise analysis of data produced a model where gender and age (P = 0.066 and P = 0.035, respectively) explained 
20% of variance of MnBin (P = 0.069) (Table V).
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Comparison of frozen and fresh blood samples
Most of the subjects (26 of 28) participating in this study had also been enrolled in a previous investigation carried out in this laboratory
5 years before (23). On that occasion, the CBMN assay had been performed on fresh whole-blood cultures. This provided the opportunity to compare the results in the CBMN assay with fresh and frozen cells from the same subjects. Mean micronucleus value and variability in fresh whole-blood sample (13.6 ± 6.5) was comparable to that recorded in the new investigation using isolated frozen lymphocytes (13.6 ± 6.3) and, furthermore, regression analysis indicated a significant correlation between the two MnBin data sets (P = 0.014, Figure 2). Taking into account, the significant influence of gender and age on spontaneous MnBin frequency, the comparison was also carried out on residual values calculated after removing the effects of these factors: in this way the correlation between MnBin in cultures from fresh and frozen cells was even more significant (P = 0.004).
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Discussion |
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In this study, the suitability of isolated frozen lymphocytes for the analysis of spontaneous and radiation-induced micronuclei in the CBMN assay was investigated. The results obtained show that following separation, freezing, cryopreservation in liquid nitrogen and thawing, human lymphocytes maintain the capacity to respond to mitogen stimulation and to produce micronuclei in cytokinesis-blocked cells.
The few studies that have focused on the investigation of the suitability of cryopreserved lymphocytes for DNA damage have not found any influence of freezing and thawing on the level of basal DNA damage evaluated by different procedures (stimulated or unstimulated lymphocytes, isolated lymphocytes or whole blood) and DNA damage assays (comet, unscheduled DNA synthesis (UDS) and cytogenetic analysis) (14–21).
Moreover our study, showing that factors affecting micronucleus yield in fresh blood cultures also modulate the incidence of micronuclei following freezing of the cells, indicates that cryopreserved lymphocytes are a valuable material and a useful alternative to fresh blood for micronucleus evaluation in population studies. In particular, gender and age of donors proved to affect MnBin also in cultures set up with frozen cells, with significantly higher incidences of micronucleated cells in females, compared with males and in aged individuals. Gender and age are recognized as the main factors affecting the yield of micronuclei in human cells, as a consequence of the preferential loss of the X chromosome (26,27). The results of this work indicate that the intrinsic cellular characteristics associated with the proness to X chromosome loss (e.g. centromere/kinetochore dysfunction) are maintained despite cell freezing and manipulation. Smoking habits did not affect the cytogenetic biomarkers analysed. On the other hand, the modifying effect of smoking in the CBMN is elusive: indeed, the majority of the studies performed, including those carried out in this laboratory (23,28), reported no association between yield of micronuclei and tobacco smoke.
Micronuclei in cytokinesis-blocked cells have been proposed as biomarkers of mutagen sensitivity in genetic susceptibility and molecular epidemiology studies (6,29). In these studies, cells are challenged with a model genotoxic agent and micronucleus yield was determined. The analysis of micronuclei may provide information on chromosome integrity and segregation, which are directly involved in cancer onset and progression. These studies might take advantage of the use of frozen cells, allowing better planning of experimental activities, confirmation of results, etc. (30). However, the experience on the use of cryopreserved cells in challenge assays is limited and contradictory, mainly concerning the analysis of DNA damage or repair in unstimulated cells (12–17). In this respect, previous studies reported contradictory results. In -irradiated or hydrogen peroxide-treated G0 lymphocytes, the same level of DNA damage evaluated by comet assay or UDS was found in cryopreserved and freshly isolated lymphocytes (14–17,21); however similar (16), lower (14,15) or higher (21) DNA repair activity was observed in the former samples. As far as the cytogenetic endpoints are concerned, after radiation treatment a significant correlation was observed in the level of breaks/cell between fresh and cryopreserved whole blood (20); conversely, micronucleus analysis in cryopreserved lymphocytes, due to high inter-experimental variability, did not allow detection of inter-individual differences or discrimination between breast cancer cases and controls (19). Even though a direct comparison between fresh and frozen cells was not feasible within this study, the results of CBMN assays with irradiated cultures indicate that isolated, frozen lymphocytes can also usefully be employed in challenge assays. The overall genotoxic effect elicited by -rays was in fact fairly similar to that observed after irradiation of fresh blood samples. Moreover, a modifying effect of age was observed on the yield of radiation-induced micronuclei, suggesting increased sensitivity to radiation in aged individuals. An increase in mutagen sensitivity within the general population was reported by some authors (13,31) and also in our laboratory (32), but not by others (33,34), and attributed to decreased DNA repair capacity in the elderly. Whatever the molecular basis of this phenomenon, the data obtained show that this character is relatively stable and maintained following freezing and long-term (>2 years) storage.
The observation that following freezing the incidence of spontaneous and radiation-induced micronuclei is modulated by the same factors affecting micronuclei in fresh cells implies the existence of stable individual susceptibility factors. As a consequence, reproducibility in micronucleus data, with intra-individual variation lower than inter-individual variation and consistency of results over time, is expected. This fact has the utmost importance if micronuclei are to be used as prognostic markers of individual cancer risk (35) and, in common with other cytogenetic biomarkers, are usually tested just once. This study provides some clues on this important aspect. The comparison of data concerning 26 individuals tested after a 5-year interval highlighted a strict intercorrelation between the two data sets, indicating that micronucleus yield can be regarded as a reliable individual descriptor of genomic stability.
In this study, a more comprehensive CBMN test protocol was applied, including the analysis of NPBs as markers of chromosome-type aberrations (dicentrics and rings) (25). NPB can provide information complementary to micronuclei, arising from events such as DNA mis-repair of strand breaks or telomere end-fusions caused by telomere shortening otherwise not detectable by scoring micronuclei only (36). Criteria for analysis and classification of NPB have recently been optimized and applied within the HUMN project (http://ehs.sph.berkeley.edu/holland/humn/). The results of this work indicate that NPB can successfully be analysed in cryopreserved samples, providing information on efficiency of recombinational DNA repair pathways following radiation challenge.
Finally, a critical aspect in most studies on human populations is represented by the amount of the blood available for biomarker analysis. In this work, it has been shown that the use of cryopreserved lymphocytes is not demanding in this respect compared to fresh blood. In fact, enough binucleated cells for the cytogenetic analyses performed were obtained using small volume (750 µl) cultures containing 0.5 x 106 to 1.0 x 106 lymphocytes/ml. Considering the loss of 20% viable lymphocytes in freezing and thawing, this means that 2.5 ml of blood may suffice to isolate and freeze lymphocytes to set up replicate cultures and carry out a mutagen sensitivity assay by the CBMN method.
In conclusion, the results of this work indicate that peripheral human lymphocytes can be successfully cryopreserved, stored and subsequently used for micronucleus analysis with the cytokinesis-block method. This technical improvement may offer significant advantages in biomonitoring and a unique chance for prospective studies on biomarkers and cancer risk.
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Acknowledgments |
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The authors are grateful to Ms Celestina D'Ascoli for her excellent and irreplaceable technical assistance and laboratory support.
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Notes |
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* To whom correspondence should be addressed at Dipartimento Ambiente e Connessa Prevenzione Primaria, Istituto Superiore di Sanità, Viale Regina Elena 299, I 00161, Rome, Italy. Tel: +39 06 49902680; Fax: +39 06 4990 3650; Email: zijno{at}iss.it
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Received on February 27, 2007; revised on April 2, 2007; accepted on April 3, 2007.
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