Mutagenesis, Vol. 17, No. 3, 211-214,
May 2002
© 2002 UK Environmental Mutagen Society/Oxford University Press
Cryopreserved versus freshly isolated lymphocytes in human biomonitoring: endogenous and induced DNA damage, antioxidant status and repair capability
2 Rowett Research Institute, Greenburn Road, Bucksburn, Aberdeen AB21 9SB, UK and 1 University of Aberdeen, Department of Biomedical Sciences, Foresterhill, Aberdeen AB25 2ZD, UK
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Lymphocytes are routinely used in human biomonitoring to assess the potential toxic and cytoprotective effects of diet on both DNA damage and repair and, by implication, health. Logistically, samples may require to be cryopreserved and stored. How this affects cells used in human biomonitoring is often not considered. In this study we have evaluated the influence of cryopreservation on endogenous and induced DNA strand breakage, altered bases (oxidized purines, oxidized pyrimidines and misincorporated uracil), antioxidant capacity and DNA repair capability in human peripheral blood lymphocytes. Neither isolation nor freezing increased DNA strand breakage above endogenous levels found in freshly isolated human lymphocytes. Oxidized bases (both pyrimidines and purines) and misincorporated uracil, were similar for fresh and frozen lymphocytes. Fresh and frozen lymphocytes responded almost identically to hydrogen peroxide. Quercetin-mediated cytoprotection against hydrogen peroxide-induced strand breakage was maintained in cryopreserved lymphocytes after short-term (24 h) and longer term (2 months) storage compared with freshly isolated and treated cells. Hydrogen peroxide-induced DNA strand breakage was repaired in fresh lymphocytes. Cryopreserved lymphocytes were unable to repair oxidant-induced DNA strand breaks. Frozen human lymphocytes can therefore be successfully used for most aspects of DNA damage biomonitoring, but not for repair.
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
There is increasing interest in the mechanisms through which diet influences DNA stability and human health. Lymphocytes provide a convenient and readily available source of human material and are routinely used experimentally to assess the potential toxic and cytoprotective effects of diet on both DNA damage and repair. Logistically, samples may require to be stored. However, how isolation, freezing and storage can affect lymphocytes used in human biomonitoring remains to be established. In a previous study we showed that endogenous DNA strand breakage was significantly increased in human lymphocytes upon storage when compared with freshly isolated cells. Moreover, contamination of lymphocyte preparations with whole blood significantly increased DNA damage (Narayanan et al., 2001
). In this study we have evaluated the effects of cryopreservation on endogenous and induced DNA strand breakage, altered bases (oxidized purines, oxidized pyrimidines and misincorporated uracil), antioxidant capacity and DNA repair capability in human peripheral blood lymphocytes.
|
Materials and methods |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
LymphoPrep lymphocyte separation medium (LSM) (specific gravity 1.077 ± 0.001 g/ml) was supplied by Nycomed (Birmingham, UK). L-Glutamine, penicillin G, sodium pyruvate and streptomycin sulphate were obtained from Sigma (Poole, UK). Dutch Modified RPMI 1640 medium was obtained from ICN Flow Laboratories (Irvine, UK). Heat-inactivated fetal calf serum (FCS) was obtained from Globepharm (Surrey, UK). Frosted microscope slides were from Richardson Supply Co. (London, UK). Low Melting Point (LMP) and High Melting Point (HMP) agarose were supplied by Gibco Life Technologies (Paisley, UK). 4',6-Diamidine-2-phenylindole dihydrochloride (DAPI) was obtained from Boehringer Mannheim (Lewes, UK). Uracil DNA glycosylase (1 U/ml) was from Helena Biosciences (Sunderland, UK). Endonuclease III and fapy glycosylase were prepared in the laboratory from over-producing plasmid vectors.
Isolation and cryopreservation of human lymphocytes
Venous blood (1x10 ml) was collected from normal healthy individuals by venepuncture (see figure legends for subject numbers). The whole blood was centrifuged at 2400 g for 15 min at 4°C, the `buffy coat' (~2 ml) removed and diluted 1:1 with RPMI and layered onto an equal volume of LSM before centrifuging at 700 g for 30 min at 20°C. The lymphocytes were transferred to a fresh centrifuge tube, washed using RPMI medium and spun for a further 15 min under the same conditions. The supernatant was decanted, the cells resuspended in RPMI containing 10% heat-inactivated FCS and counted using a Neubauer Improved Haemocytometer. Isolated lymphocytes were either used (or treated) immediately (freshly isolated lymphocytes) or were centrifuged and resuspended in 0.5 ml aliquots at 3x106 cells/ml in freezing mix (90% v/v heat-inactivated FCS and 10% v/v DMSO), frozen at –1°C/min in polystyrene and stored at –80°C. For analysis of DNA damage, antioxidant status and repair capability, the aliquots were rapidly thawed at 37°C, immediately centrifuged at 200 g for 3 min at 4°C to remove the freezing mix and the lymphocyte pellet resuspended gently in 0.4 ml of RPMI + 10% (v/v) heat-inactivated FCS. Aliquots (40 µl) of this lymphocyte suspension were used for subsequent Comet analysis.
Endogenous DNA strand breakage, oxidized base damage (pyrimidines and purines) and misincorporated uracil levels in fresh and cryopreserved lymphocytes measured using the Comet assay
Cells were suspended in 85 µl of 1% (w/v) LMP agarose in phosphate-buffered saline (PBS), pH 7.4, at 37°C and immediately pipetted onto a frosted glass microscope slide precoated with a layer of 1% (w/v) HMP agarose similarly prepared in PBS. The agarose was allowed to set for 5 min at 4°C and the slide incubated in lysis solution (2.5 M NaCl, 10 mM Tris, 100 mM Na2EDTA, NaOH to pH 10.0 and 1% v/v Triton X-100) at 4°C for 1 h to remove cellular proteins. After lysis, the slides were washed three times for 5 min each in uracil DNA glycosylase buffer (60 mM Tris–HCl, 1 mM EDTA, 0.1 mg/ml BSA, pH 8.0) or endonuclease III/fapy glycosylase buffer (40 mM HEPES–KOH, 0.1 M KCl, 0.5 mM EDTA, 0.2 mg/ml BSA, pH 8.0) and blotted with tissue paper. The gel was covered with either 50 µl of appropriate buffer (strand breakage), with uracil DNA glycosylase (misincorporated uracil) or with endonuclease III (oxidized pyrimidines) or fapy glycosylase (oxidized purines), sealed with a coverslip and incubated at 37°C for up to 1 h (see figure legends) in a moist atmosphere. The slides were subsequently aligned in a 260 mm wide horizontal electrophoresis tank containing buffer (1 mM Na2EDTA and 0.3 M NaOH, pH 12.7) for 40 min before electrophoresis at 25 V for 30 min (at an ambient temperature of 4°C with the temperature of the running buffer not exceeding 15°C). The slides were washed three times at 4°C for 5 min each with neutralizing buffer (0.4 M Tris–HCl, pH 7.5) before staining with 20 µl of DAPI (5 µg/ml) (Duthie and McMillan, 1997).
Hydrogen peroxide-induced DNA strand breakage in fresh and cryopreserved lymphocytes
Lymphocytes (either freshly isolated or cryopreserved then thawed) were incubated in plastic tubes with hydrogen peroxide (200 µM in PBS for 5 min on ice), washed and resuspended in LMP agarose for analysis of DNA strand breakage by the Comet assay.
Quercetin-mediated cytoprotection against oxidative DNA damage in fresh and cryopreserved lymphocytes
Freshly isolated lymphocytes (1x106/ml) were preincubated in RPMI medium containing 10% (v/v) heat-inactivated FCS, 100 U/ml penicillin, 100 µg/ml streptomycin, 2 mM L-glutamine and 100 µg/ml sodium pyruvate in sterile 25 cm3 tissue culture flasks either with quercetin (50 µM in DMSO) or DMSO for 2 h at 37°C in 5% CO2/95% air. An aliquot of lymphocytes (50 µl) was taken from each flask, washed once with PBS, exposed to hydrogen peroxide (200 µM in PBS for 5 min on ice) as before and resuspended in LMP agarose for analysis of DNA strand breakage by the Comet assay. The remaining cultured cells were recovered from the flasks, washed once with PBS and cryopreserved as described previously (see above). The frozen lymphocytes were recovered, thawed and treated with hydrogen peroxide, followed by Comet analysis, exactly as described for fresh lymphocytes, after 24 h and 2 months storage at –80°C.
DNA repair in fresh and cryopreserved lymphocytes
To induce DNA damage, human lymphocytes (fresh or cryopreserved) were washed once in PBS before exposure to H2O2 (200 µM in PBS on ice for 5 min). The cells were either resuspended immediately in LMP agarose for detection of DNA strand breakage by Comet analysis or were incubated in plastic tubes at 37°C in 95% air/5% CO2 in complete culture medium for up to 24 h to determine repair of oxidative damage.
Quantitation of the Comet assay
Nucleoids were scored visually using a Zeiss Axioskop fluorescence microscope. One hundred comets from each slide (scored at random) were classified into one of five classes according to the relative intensity of fluorescence in the tail and given a value of 0–4 (from undamaged, 0, to maximally damaged, 4). The total score for 100 comets can range from 0 (all undamaged) to 400 (all maximally damaged) and is expressed in arbitrary units. DNA strand breakage was estimated based only on the score obtained from buffer-treated gels. Misincorporated uracil or oxidized pyrimidines and purines are measured by subtracting the visual score obtained for buffer-treated gels from the score obtained after incubation with appropriate enzyme. This method of visual classification has been extensively validated by comparison with comets selected using computerized image analysis (Duthie and McMillan, 1997).
Statistical analysis
Student's t-test and ANOVA, together with the Tukey's honestly significant difference test, were carried out as appropriate using SPSS 8.0 for Windows.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Potential detrimental effects of the process of isolation from whole blood on lymphocyte DNA strand breakage (by centrifugation through a Percol density gradient) was investigated. Neither the isolation nor the freezing process increased DNA strand breakage above endogenous levels found in fresh human lymphocytes (26.0 ± 1.1 in fresh cells, compared with 26.7 ± 1.6 in freshly isolated cells and 26.8 ± 1.3 in cryopreserved and thawed lymphocytes for n = 6 in all cases).
The levels of oxidized bases (both pyrimidines and purines) and misincorporated uracil were similar for fresh and frozen lymphocytes (Table I). Moreover, fresh and frozen lymphocytes responded almost identically (in terms of DNA strand breakage) to exposure to hydrogen peroxide (Table I).
|
Quercetin-mediated cytoprotection against hydrogen peroxide-induced strand breakage was maintained in cryopreserved lymphocytes after short-term (24 h) and longer term (2 months) storage compared with freshly isolated and treated cells (Figure 1
).
|
Hydrogen peroxide-induced DNA strand breakage was progressively and efficiently repaired to approximately endogenous levels in freshly isolated lymphocytes (Figure 2
). Conversely, cryopreserved lymphocytes were unable to repair DNA strand breaks following exposure to hydrogen peroxide (Figure 2). Moreover, incubation at 37°C induced DNA damage in both untreated and hydrogen peroxide-exposed frozen lymphocytes (Figure 2).
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
In this study we wanted to determine whether frozen lymphocytes are a viable alternative to freshly isolated lymphocytes for the study of aspects of DNA damage and repair. The effects of cryopreservation on endogenous and induced DNA strand breakage, altered bases (oxidized purines, oxidized pyrimidines and misincorporated uracil), antioxidant capacity following incubation with the dietary flavonoid quercetin and DNA repair capability were measured in human peripheral blood lymphocytes.
Single cell gel electrophoresis (SCGE, known as the Comet assay) is a rapid, sensitive and reliable method of measuring DNA strand breaks in individual cells (McKelvey-Martin et al., 1993). Breaks in the DNA cause relaxation of the DNA, which are visualized during electrophoresis. Comets are detected by fluorescence microscopy after staining with various fluorescent dyes. The intensity of fluorescence in the tail is proportional to the frequency of DNA strand breaks (Olive et al., 1990).
The Comet assay is commonly used in human biomonitoring to measure DNA strand breakage (Kassie et al., 2000). Detrimental effects of pollutants, environmental contaminants and occupational exposure on DNA stability and the mechanisms associated with the cytoprotective properties of the human diet have been assessed using SCGE (Kassie et al., 2000). The usefulness of the Comet assay in biomonitoring has been expanded significantly by the inclusion of bacterial DNA repair enzymes which specifically detect several types of altered bases, such as oxidized pyrimidines, oxidized purines and misincorporated uracil (Collins et al., 1993; Duthie and McMillan, 1997). Endogenous oxidative base damage in human lymphocytes is decreased following supplementation with either an antioxidant cocktail of ß-carotene, vitamin C and vitamin E (Duthie et al., 1996) or more complex food sources such as carrot juice (Pool-Zobel et al., 1997), onions or tea (Lean et al., 1999; Boyle et al., 2000).
Poor folate intake has been implicated in the development of colorectal cancer (Kim and Mason, 1997). Misincorporation of uracil as a result of folate deficiency may act to destabilize DNA. Both human lymphocytes and colonocytes made folic acid deficient have increased uracil levels, as measured using a modified Comet assay (Duthie and McMillan, 1997; Duthie et al., 2000). It is important, therefore, to determine whether isolation, freezing and thawing negatively modify either endogenous DNA strand breakage or base damage in human lymphocytes. Cryopreservation has no detrimental effect on either endogenous or mutagen-induced sister chromatid exchange frequency or on colony-forming ability and HPRT mutation frequency in human lymphocytes (Murli et al., 1987; Norimura et al., 1990; Cheng et al., 2001). In this study the levels of oxidized pyrimidines, oxidized purines and uracil were similar in both freshly isolated and frozen lymphocytes, indicating that the DNA damage that is already present in the DNA of fresh cells is maintained throughout the isolation and storage procedure.
Several human studies have investigated the possible protective effects of dietary antioxidants such as lycopene, ß-carotene, vitamin C, vitamin E and quercetin, by measuring the ex vivo resistance of lymphocytes to oxidative attack (Duthie et al., 1996; Jenkinson et al., 1999; Riso et al., 1999; Boyle et al., 2000). One potential confounding factor when assessing the results from these types of studies is the influence that isolation, freezing and thawing might have on the antioxidant capacity of the cells. The response of cryopreserved lymphocytes to hydrogen peroxide treatment (DNA strand breakage) was not significantly different from that in fresh cells in this study. To further test whether cryopreservation altered the antioxidant capacity of frozen lymphocytes and their subsequent ability to resist oxidative attack, human lymphocytes were loaded with the dietary flavonoid quercetin prior to freezing. The level of quercetin-mediated cytoprotection was similar whether lymphocytes were treated fresh or had been frozen for 24 h or 2 months. Pretreatment with quercetin resulted in a 22–29% decrease in oxidant-induced DNA strand breakage. It appears in this study that the response to oxidative attack and the antioxidant capacity of frozen lymphocytes is maintained at least for 2 months following isolation, storage and thawing. Flavonoids become only weakly associated with the cell membrane following uptake, suggesting that the antioxidant ability of other nutrients, such as the lipid-soluble carotenoids, which become integrated into the cell membrane, and water-soluble vitamin C, may also be maintained during isolation and cryopreservation.
DNA repair has a profound influence on DNA stability and ultimately cancer incidence. It remains to be determined in human studies how diet and nutrition might influence DNA repair capacity in individuals and whether this is related to subsequent cancer risk. In this study, cryopreserved lymphocytes were unable to repair hydrogen peroxide-induced DNA strand breaks when compared with freshly isolated cells. Moreover, incubating frozen cells at 37°C for >4 h caused a dramatic increase in both induced and endogenous DNA damage. This is in agreement with Visvardis et al. (1997), who found that cryopreserved human lymphocytes were unable to repair hydrogen peroxide-induced DNA strand breakage after 2 h in culture. In contrast to our findings, no difference in endogenous DNA damage between the two cell types was reported (Visvardis et al., 1997). However, in this study, lymphocytes were incubated for a more extended period, i.e. up to 24 h. Cell viability (membrane damage measured by trypan blue exclusion) decreases in lymphocytes incubated for 24 h at 37°C (Capelli et al., 1982). DNA stability was not measured in that study. Quiescent lymphocytes have low cellular deoxynucleotide pools and poor efficiency of excision break rejoining, making them more sensitive to certain mutagens when compared with mitogen-stimulated lymphocytes (Green et al., 1994). While DNA repair can be readily investigated in cultured lymphocytes that have been stimulated to divide (Green et al., 1994; Visvaradis et al., 1997) it remains to be established if this is really an appropriate way to measure the potential influence of diet on individual differences in DNA repair capacity. DNA repair activity may be higher in stimulated cryopreserved lymphocytes following freezing and thawing compared with fresh cells due to selective survival of lymphocytes with relatively high DNA repair activity (Risom and Knudsen, 1999). Moreover, culturing lymphocytes may induce DNA damage above background levels as a result of sudden exposure to atmospheric oxygen (Torbergsen and Collins, 2000). Measuring the repair capacity of a subcellular extract of human lymphocytes in vitro may prove a more simple and relevant way of assessing DNA repair in a human population (Collins et al., 2001).
These results indicate that peripheral human lymphocytes can be successfully cryopreserved, stored and subsequently used for some methods of determining DNA damage in human biomonitoring. Endogenous levels of DNA strand breaks and altered bases appear to be maintained during both isolation and freezing, while the response of the cells to induced oxidative DNA damage is similar for fresh and frozen cells. Moreover, the cellular antioxidant environment of frozen lymphocytes is preserved over several months, with the subsequent responses to oxidative challenge being similar for fresh and frozen cells. Conversely, cryopreservation reduces the capacity for DNA excision repair in non-mitogen-stimulated frozen lymphocytes and actually decreases DNA integrity in these cells. Whether this is due to leakage of essential DNA repair components from the cell as a result of freezing and thawing-induced physical changes in the membrane or diminished enzyme lability throughout isolation and storage remains to be established.
|
Acknowledgments |
|---|
This work was funded by the Scottish Executive Environment and Rural Affairs Department (SEERAD) and the World Cancer Research Fund (WCRF).
|
Notes |
|---|
2 To whom correspondence should be addressed. Tel: +44 1224 712751; Fax: +44 1224 716629; Email: sd{at}rri.sari.ac.uk
|
References |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
-
Boyle,S.P., Dobson,V.L., Duthie,S.J., Kyle,J.A.M. and Collins,A.R. (2000) Absorption and DNA protective effects of flavonoid glycosides from an onion meal. Eur. J. Nutr., 39, 213–223.[ISI][Medline]
Capelli,E., Stefanini,M., Giulotto,E. and Nuzzo,F. (1982) Validation of a DNA repair synthesis assay on pools of fresh and frozen-thawed lymphocytes. Mutat. Res., 104, 187–191.[ISI][Medline]
Cheng,L., Wang,L.E., Spitz,M.R. and Wei,Q. (2001) Cryopreserving whole blood for functional assays using viable lymphocytes in molecular epidemiology studies. Cancer Lett., 166, 155–163.[ISI][Medline]
Collins,A.R., Duthie,S.J. and Dobson,V.L. (1993) Direct enzymic detection of endogenous oxidative base damage in human lymphocyte DNA. Carcinogenesis, 14, 1733–1735.
Collins,A.R., Dusinska,M., Horvathova,E., Munro,E., Savio,M. and Stetina,R. (2001) Inter-individual differences in repair of DNA base oxidation, measured in vitro with the Comet assay. Mutagenesis, 16, 297–301.
Duthie,S.J. and McMillan,P. (1997) Uracil misincorporation in human DNA detected using single cell gel electrophoresis. Carcinogensis, 18, 1709–1714.
Duthie,S.J., Ma,A.-G., Ross,M.A. and Collins,A.R. (1996) Antioxidant supplementation decreases oxidative DNA damage in human lymphocytes. Cancer Res., 56, 1291–1295.
Duthie,S.J., Narayanan,S., Blum,S., Pirie,L. and Brand G. (2000) Folate deficiency in vitro induces uracil misincorporation and DNA hypomethylation and inhibits DNA excision repair in immortalised normal human colon epithelial cells. Nutr. Cancer, 37, 245–251.[ISI][Medline]
Green,M.H., Waugh,A.P., Lowe,J.E., Harcourt,S.A., Cole,J. and Arlett,C.F. (1994) Effect of deoxyribonucleosides on the hypersensitivity of human peripheral blood lymphocytes to UV-B and UV-C irradiation. Mutat. Res., 315, 25–32.[ISI][Medline]
Jenkinson,A.McE., Collins,A.R., Duthie,S.J., Wahle,K.W.J. and Duthie,G.G. (1999) The effect of increased intakes of polyunsaturated fatty acids and vitamin E on DNA damage in human lymphocytes. FASEB J., 13, 2138–2142.
Kassie,F., Parzefall,W. and Knasmuller,S. (2000) Single cell gel electrophoresis: a new technique for human biomonitoring studies. Mutat. Res., 463, 13–31.[ISI][Medline]
Kim,Y.-I. and Mason,J.B. (1997) Folate, epithelial dysplasia and colon cancer. Proc. Assoc. Am. Phys., 107, 218.
Lean,M.E.J., Noroozi,M., Kelly,I., Burns,J., Talwar,D. and Sattar,N. (1999) Dietary flavonols protect diabetic human lymphocytes against oxidative damage to DNA. Diabetes, 48, 176–181.[Abstract]
McKelvey-Martin,V.J., Green,M.H.L., Schmezer,P., Pool-Zobel,B.L., De Méo,M.P. and Collins,A.R. (1993) The single cell gel electrophoresis assay (comet assay): a European review. Mutat. Res., 288, 47–63.[ISI][Medline]
Murli,H., Galloway,S.M., Ivett,J.L., Parry,D.M. and Mulvihill,J.J. (1987) Baseline and mutagen-induced sister-chromatid exchanges in cultures of human whole blood and purified fresh or frozen lymphocytes. Mutat. Res., 180, 101–108.[ISI][Medline]
Narayanan,S., O'Donovan,M.R. and Duthie,S.J. (2001) Lysis of whole blood in vitro causes DNA strand breaks in human lymphocytes. Mutagenesis, 16, 455–459.
Norimura,T., Maher,V.M. and McCormick,J.J. (1990) A quantitative assay for measuring the induction of mutations in human peripheral blood lymphocytes. Mutat. Res., 230, 101–109.[ISI][Medline]
Olive,P.L., Banath,J.P. and Durand,R.E. (1990) Heterogeneity in radiation-induced DNA damage and repair in tumour and normal cells measured using the `Comet assay'. Radiat. Res., 122, 86–94.[ISI][Medline]
Pool-Zobel,B.L., Bub,A., Muller,H., Wollowski,I. and Rechkemmer,G. (1997) Consumption of vegetables reduces genetic damage in humans: first results of a human intervention trial with carotenoid-rich foods. Carcinogenesis, 18, 1847–1850.
Riso,P., Pinder,A., Santangelo,A. and Porrini,M. (1999) Does tomato consumption effectively increase the resistance of lymphocyte DNA to oxidative damage? Am. J. Clin. Nutr., 69, 712–718.
Risom,L. and Knudsen,L.E. (1999) Use of cryopreserved peripheral mononuclear blood cells in biomonitoring. Mutat. Res., 440, 131–138.[ISI][Medline]
Torbergsen,A.C. and Collins,A.R. (2000) Recovery of human lymphocytes from oxidative DNA damage; the apparent enhancement of DNA repair by carotenoids is probably simply an antioxidant effect. Eur. J. Nutr., 39, 80–85.[ISI][Medline]
Visvardis,E.-E., Tassiou,A.M. and Piperakis,S.M. (1997) Study of DNA damage induction and repair capacity of fresh and cryopreserved lymphocytes exposed to H2O2 and 
-irradiation with the alkaline comet assay. Mutat. Res., 383, 71–80.[ISI][Medline]
Received on November 19, 2001; accepted on January 2, 2002.
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  M. Dusinska and A. R. Collins The comet assay in human biomonitoring: gene-environment interactions Mutagenesis, May 1, 2008; 23(3): 191 - 205. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Zijno, F. Saini, and R. Crebelli Suitability of cryopreserved isolated lymphocytes for the analysis of micronuclei with the cytokinesis-block method Mutagenesis, September 1, 2007; 22(5): 311 - 315. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 X. Zhao, G. Aldini, E. J Johnson, H. Rasmussen, K. Kraemer, H. Woolf, N. Musaeus, N. I Krinsky, R. M Russell, and K.-J. Yeum Modification of lymphocyte DNA damage by carotenoid supplementation in postmenopausal women Am. J. Clinical Nutrition, January 1, 2006; 83(1): 163 - 169. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Z. Wei, J. Lifen, H. Jiliang, L. Jianlin, W. Baohong, and D. Hongping Detecting DNA repair capacity of peripheral lymphocytes from cancer patients with UVC challenge test and bleomycin challenge test Mutagenesis, July 1, 2005; 20(4): 271 - 277. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||