- Email: [email protected]
Use of cryopreserved peripheral mononuclear blood cells in biomonitoring
Mutation Research 440 Ž1999. 131–138
Use of cryopreserved peripheral mononuclear blood cells in biomonitoring Lotte Risom b
a,)
, Lisbeth E. Knudsen
b
a Danish Cancer Society, StandbouleÕarden 49, DK-2100 Copenhagen Ø, Denmark National Institute of Occupational Health, Lersø Park Alle´ 105, DK-2100 Copenhagen Ø, Denmark
Received 2 October 1998; received in revised form 2 February 1999; accepted 4 February 1999
Abstract This study was performed to investigate the effect of storing blood samples by freezing on selected biomarkers and possible implications for biomonitoring. Comparative measurements were performed in order to investigate the use of cryopreserved vs. freshly separated peripheral mononuclear blood cells ŽPMBC. obtained from donor blood. Measurements of DNA-repair, mutant frequency, and subcell content were included. Samples for large biomonitoring studies are usually taken from study groups within a short timeperiod of daysrweeks and storing of study material for later analysis can be necessary. We measured the DNA repair activity as dimethylsulfate induced unscheduled DNA synthesis ŽUDS. in PMBC incubated with either autologous plasma or fetal bovine serum ŽFBS.. Comparison of the hprt mutant frequency by the T cell cloning assay was made in parallel. Finally the content of BrT-lymphocytes and monocytes was measured in phytohemaglutinin ŽPHA.-stimulated cultures at different time intervals. The results showed a higher DNA repair activity in cryopreserved samples compared with fresh samples. We also found differences in mutant frequencies with higher values in fresh samples. A significant correlation of frequencies was seen when comparing fresh with cryopreserved samples. Furthermore we recommend fresh human plasma used in UDS incubation media. q 1999 Elsevier Science B.V. All rights reserved. Keywords: Biomonitoring; Peripheral mononuclear blood cell; Cryopreservation; Storing
1. Introduction In large biomonitoring studies sampling logistics often puts demands for the storing of sample material for later analysis. First of all it is usually not possible to analyse all the samples within the study group on the day of sampling, because of the hours spent during the ) Corresponding author. Fax: q45-35-25-77-31; E-mail: [email protected]
collection of materials and the processing of samples and assaying due to time-consuming methods. Secondly seasonal variation of individual DNA damage and DNA repair has been reported. During summertime the sunlight exposure is high because of sunnier weather, increased outdoor life and less covering of the skin. Also changes in lifestyle, e.g., food consumption may account for seasonal variations. An association in the level of DNA damage and DNA repair with season in Denmark has indicated an association with sunlight exposure w1x. Also a
1383-5718r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved. PII: S 1 3 8 3 - 5 7 1 8 Ž 9 9 . 0 0 0 1 9 - 4
132
L. Risom, L.E. Knudsenr Mutation Research 440 (1999) 131–138
seasonal variation in measurements of micronuclei and sister chromatid exchange with higher levels in summertime has been reported w2x. Another study found a positive association between hprt mutant frequency in peripheral mononuclear blood cells ŽPMBC. and sunlight records in the United Kingdom w3x. The influence of seasonal variation on the sample material should be taken into account when comparing data. To minimise any seasonal variation between study persons, samples should therefore preferably be collected during a short period of time. Freezing blood cells may however introduce changes in the content of different subpopulations of white blood cells, due to heterogeneity in survival in the cell population as a result of the freezingrthawing process of cryopreserved samples. Changes in the proportion of subcells after storage could contribute to differences in, e.g., DNA repair activity and mutant frequency. Studies have reported a higher DNA repair activity in monocytes and B-lymphocytes compared to T-lymphocytes w4x. Other studies have revealed heterogeneity, e.g., a higher level of adducts in smokers’ lymphocyte DNA compared with granulocyte DNA w5x. Furthermore, freezing andror thawing may introduce DNA damage or mutations from endogenous processes in the cell. Therefore, one may not be able to compare results from fresh and cryopreserved samples. With the need in the nearby future, for storing large amounts of samples in biomonitoring studies for later analysis, we have investigated the impact of freezing blood samples. To evaluate the effect of cryopreservation we examined the DNA repair activity measured by dimethylsulfate ŽDMS. induced unscheduled DNA synthesis ŽUDS.. The T cell cloning assay was used in measurements of mutations in fresh and frozen PMBCs. Changes in cell population were determined in subpopulations of blood cells at different times of growing. UDS and the T cell cloning are both assays used in biomonitoring as measurements of individual environmental and occupational exposure. Measure of UDS has been used in studies of vulcanizers, stainless steel welders and sun exposure w6,7,1x. UDS is considered an indicator of cancer susceptibility following the important role of DNA repair in carcinogenesis. A low DNA repair is associated with higher cancer risk. Several studies have measured mutational effect in the hprt gene in
relation to exposure to diesel exhaust in bus drivers, to smoking, and to styrene in lamination workers w8–10x. Cryopreservation of PMBC samples for later analysis is a common and convenient procedure in biomonitoring studies, with measurements of, e.g., 32 P-postlabelling, host cell reactivation assay and T cell cloning w11–13x, while no studies of UDS have been found measured in cryopreserved samples.
2. Materials and methods 2.1. Cell biology All cell culture media, fetal bovine serum ŽFBS. and antibiotics were from Gibco BRL. The blood banks at Bispebjerg Hospital and Rigshospitalet, Copenhagen kindly supplied buffy coats and human serum ŽHS.. Dimethylsulfoxide ŽDMSO. and dimethylsulfate ŽDMS. were from Merck and Fluka respectively. Hydroxyurea ŽHU., phytohemaglutinin ŽPHA. and 6-thioguanine ŽTG. were from Sigma. g-Irradiation of lymphocytes was carried out at Department of Clinical Immunology, Rigshospitalet, Copenhagen. 2.2. Isolation of peripheral mononuclear blood cells Buffy coats from healthy donors were obtained from approximately 500-ml venous blood. Isolation of the peripheral mononuclear blood cells was done by gradient centrifugation 30 min at 800 = g using Lymphoprep w ŽNycomed Pharma AS, r s 1.077.. The autologous plasma was recovered from the upper phase of the separated cell layer. The mononuclear cells were isolated from the mid-layer and washed twice in cold 1 = PBS before suspension in basic medium ŽBM.. 2.3. Culture media BM is RPMI 1640 supplemented with 25 mM hepes, 100 IUrml benzylpenicilium and 100 mgrml streptomycin. Nutrient Medium ŽNM. is BM with 5% heat inactivated FBS and 5% HS. Growth medium ŽGM. is NM with 20% AIM V Žserum-free lymphocyte medium used for culturing functionally
L. Risom, L.E. Knudsenr Mutation Research 440 (1999) 131–138
differentiating lymphoid cell lines., 1 mgrml PHA and 20% T-cell growth factor enriched Conditioned Medium ŽCM. w14x. Both CM and HS were tested for the ability to promote long-term proliferation of activated T-cells in microplates and stored at y208C. 2.4. Freezing and thawing of cells Cells for freezing were adjusted to 1 = 10 7 cellsrml in cold BM containing 50% FBS and 10% DMSO. Immediately after suspension in media cells were transferred to cold cryotubes, stored in isopropanol containers at y708C Ž18Crmin. for 24 h before being transferred for storing at liquid nitrogen. Thawing of cells was done fast by incubation at 378C in water bath, gently turning the tubes a few times. When 9r10 of the cell suspension was thawed it was slowly mixed with 9 ml cold media containing 10% wrw dextrose ŽDifco, Detroit MI, USA., 40% RPMI 1640 and 50% FBS, before centrifugation at 800 = g for 10 min. The cells were kept on ice bath prior to analysis. 2.5. Unscheduled DNA synthesis The UDS protocol was modified from w15,16x. UDS is an indirect measure of overall DNA repair of induced damage. The isolated peripheral mononuclear blood cells were counted in haemacytometer and adjusted to 2 = 10 6 cellsrml in BM. For in vitro damage 2 = 10 6 cells, were incubated with 0 or 10 mM DMS for 30 min at 378C. After centrifugation DMS was removed and the cells resuspended in BM. The UDS was measured by incorporation of w3 Hxthymidine into DNA. 2 = 10 5 cells were dissolved in BM with 20% autologous plasma, 20% HS or 20% FBS and incorporated with 0.4 mM w3 Hxthymidine Ž25 Cirmmol, Amersham. at 378C for 18 h in microtiter wells Ž96 wells, 7 mm round bottom, Nunc.. Background UDS was determined in parallel cultures exposed to DMSO. To minimise replication of DNA, the cells were incubated with 10 mM of HU. The measurements were performed in replicates of eight in 96 microtiter wells. The cells were harvested in a cell harvester ŽMach 96, Wallac. transferring the washed cells onto a glass fibre filter, which was counted in a betaplate scintillation counter. The UDS results are given as cpm w3 Hxthymidine
133
incorporated into 2 = 10 5 cells, exposed to DMS, background subtracted. 2.6. T cell cloning The T cell cloning assay was performed as described by w17x with modifications made by w14,18x. After washing twice in cold PBS lymphocytes were resuspended in cold NM at 1 = 10 6 cellsrml. Cells were then incubated at 378C with 44 mgrml PHA for 44 h. Two non-selective plates were prepared by inoculating 2 target cells and 2 = 10 4 freshly g-irradiated Ž40 Gy. lymphoblastoid RJK853 cells Žkindly provided by Dr. Hou at Environmental Medicine Unit, Karolinska Institute, CNTrNOVUM, Huddinge, Sweden. as feeder cells in 200 ml GM in each well. Selection plates received 2 = 10 4 target cells and 1 = 10 4 feeder cells per well containing 2 mgrml TG Ž12 mM.. A stock solution of 1 mgrml of TG in 0.01 M NaOH was made immediately before use. The plates were wrapped in plastic foil to avoid evaporation and incubated at 378C in 5% CO 2 in air at 95% humidity for 2 weeks without medium change. Wells were then scored visually for the presence of clones in the wells using an inverted microscope. After 3–4 days the plates were inspected again to check whether slowly growing clones had been missed. Cloning efficiency ŽCE. with and without TG was calculated from the proportion of negative wells Ž P0 . assuming a Poisson distribution of clone forming cells between wells, i.e., P0 s eyx , and CE s xrn, where n s number of cells seeded per well. The frequency of mutant cells ŽMF. was given by CE in the presence of TG divided by the CE in the absence of TG w17x. 2.7. Quantificationr distribution of PMBC content Quantification of content of B- and T-lymphocytes and monocytes in peripheral mononuclear blood cell fractions was measured by incubation with PHA for 0, 24, 48, 72 and 96 h. Samples of PMBC was incubated with specific monoclonal antibodies towards T-lymphocytes ŽAnti-Leu-4-CD3. and Blymphocytes Ž Anti-Leu-12-CD19 . Ž Simultest CD3rCD19, Becton Dickinson.. A separate cell sample was incubated with antibodies towards
L. Risom, L.E. Knudsenr Mutation Research 440 (1999) 131–138
134
monocytes ŽAnti-Leu-M3-CD14, PE Becton Dickinson.. The monoclonal antibodies are conjugated to different fluorochromes: fluorescein conjugate or phycoerythrin conjugate. When antibodies bound, cells are excited by a single light source from a fluorescence microscope, T-cells display green fluorescence Žfluorescin. and B-cells orange-red fluorescence Žphycoerythrin.. 5 = 10 5 cells were incubated with 10 ml antibodies for 30 min on ice in a total volume of 20 ml. Slides were prepared using 10 ml of the cell suspension. The slide was covered with coverslip, protected from light and allowed to stand for 2 min. The contents of green and orange fluorescing cells and the total number of cells were counted at 40 = in the epi-fluorescence microscope. To count the total amount of cells the microscope was switched from epi-fluorescence to transmission of visible light. The number of B- and T-cells and the total number of cells were counted Žminimum 100 cells.. In the separate sample staining monocytes the number of monocytes and the total number of cells were counted. The subcell populations are calculated as percentage of cells in the sample.
2.8. Statistics The means, standard deviations and correlations were calculated by use of the SAS computer program. The UDS measurements were compared by analysis for significant differences among mean values by the non-parametric Mann–Whitney statistical analysis. The Student’s t-test was used to compare the cloningrmutant frequency of cryopreserved vs. fresh samples. The Pearson correlation analysis was performed to test for systematic differences, e.g., higher levels in fresh compared with cryopreserved samples. The level of significance was 5%.
3. Results 3.1. UDS in primary human PMBCs A total of 19 healthy persons were used to measure background UDS and DNA repair activity of DMS induced UDS minus background UDS of PMBCs obtained from buffy coats ŽTable 1.. Due to
Table 1 DNA repair in fresh and cryopreserved PMBCs using autologous plasma in incubation media Donor code
Fresh samples
Cryopreserved samples
DMSO Žbackground.
DMS Ž10 mM. induced UDS
DNA repair ŽDMS–DMSO.
DMSO Žbackground.
DMS Ž10 mM. induced UDS
a DNA repair ŽDMS–DMSO.
39 40 71 06 50 98 58 59 940 941 942 960 961 962
137b 126 182 107 68 107 191 156 256 266 242 108 133 174
288 252 155 193 92 174 205 231 257 295 313 136 106 239
151 126 y27 86 24 67 14 75 1 29 71 28 y27 65
136 238 169 132 213 322
325 607 255 333 321 576
189 369 86 201 108 254
c
c
c
c
c
c
142 198 153 202 259 171
198 331 291 270 346 216
56 133 138 68 87 45
Mean " S.D.
161 " 61
210 " 70
49 " 53
195 " 57
339 " 127
145 " 95
a
a
DNA repair activity is DMS induced UDS minus DMSO background. Result given as cpm w3 Hxthymidine incorporated into 2 = 10 5 cells. c Samples where only autologous plasma for fresh samples could be collected. b
L. Risom, L.E. Knudsenr Mutation Research 440 (1999) 131–138 Table 2 Cloning efficiency and mutant frequency of 22 individual fresh and cryopreserved donor samples Donor code
Fresh samples
Cryopreserved samples y6
CEr%
MF=10
CEr%
MF=10y6
54 59 61 65 85 92 39 40 31 35 71 04 83 84 88 98 63 64 78 80 064 088
28 19 30 37 34 24 44 40 16 36 49 44 10 22 36 37 37 47 28 31 12 12
10.3 4.1 15.8 11.8 11.2 30.3 8.6 4.7 32.0 6.1 5.2 12.7 19.5 8.2 8.8 5.4 6.4 7.9 6.6 9.2 39.7 36.7
21 39 40 23 37 24 49 50 33 21 26 37 31 26 25 18 28 27 20 35 26 24
7.6 12.9 13.3 9.3 3.3 39.0 4.2 1.7 7.8 4.3 1.1 7.8 7.2 10.3 10.7 12.1 5.7 12.2 9.6 6.3 18.5 17.1
Mean"S.D.
31"12
13.7"10.9
30"9
10.0"7.7
135
incubated with autologous plasma showed significantly higher values in cryopreserved samples p s 0.004, n s 12 ŽTable 1.. Additionally, samples with FBS in incubation media showed significantly higher values in cryopreserved samples p s 0.002, n s 12 Ždata not shown.. In fresh samples significantly higher UDS values were found in samples incubated with autologous plasma compared with samples incubated with FBS p s 0.01, n s 14 Žwhereas in frozen samples p s 0.22, n s 8 when comparing incubation media.. Values obtained by use of autologous plasma were tested for correlation with values of FBS in incubation medium. A significant correlation was found with r s 0.58, p s 0.03, n s 14 and r s 0.92, p s 0.001, n s 8 for fresh and cryopreserved samples, respectively.
3.2. Mutant frequency in PMBCs
CE, cloning efficiency; MF, mutation frequency; S.D., standard deviation.
lack of induced repair some samples exhibited negative values. FBS was tested as enrichment in medium as substitute for autologous plasma, which was only available for 14 fresh samples and 12 cryopreserved samples. The results of background and DNA repair activity UDS in fresh and cryopreserved samples
A total of 22 donor samples could be used in the statistical calculations of both CE and MF between pair measurements of individual fresh and cryopreserved samples ŽTable 2.. MF in fresh samples yielded mean of 13.7 " 10.9 Ž=10y6 . and cryopreserved samples yielded mean MF of 10.0 " 7.7 Ž=10y6 .. The Student’s t-test revealed significant difference Ž p s 0.001.. To test for association between fresh and cryopreserved samples of individual MF measurements the Pearson correlation was calculated and found significant Ž r s 0.58, p s 0.004, n s 22., which implies association. Fresh samples yielded mean CE of 31% " 12 and cryopreserved samples yielded mean CE of 30% " 9 after cloning. No difference was seen Ž p s 0.82..
Table 3 Variation in concentrations of monocytes, B-and T-cells in PHA stimulated PMBCs at different times during incubation PHArhour a
0 24 48 72 96 a
N
8 6 8 8 6
Freshly isolated PMBC
N
%B
%T
%Mono
12 8 10 8 6
49 64 56 70 86
34 26 20 21 11
6 3 6 6 4
Cryopreserved PMBC %B
%T
%Mono
12 8 5 9 7
70 86 84 79 79
27 10 15 16 16
Phytohemaglutinin ŽPHA. stimulation per hour. N, Number of samples; PMBC, peripheral mononuclear blood cell; B, B-cells; T, T-cells; Mono, monocytes.
136
L. Risom, L.E. Knudsenr Mutation Research 440 (1999) 131–138
3.3. Variation in subgroups of PMBCs The content of monocytes, T- and B-cells were measured just before incubation Ž t s 0. and after 24 Ž t s 24., 48 Ž t s 48., 72 Ž t s 72. and 96 Ž t s 96. h of PHA-incubation. Measurements of variation in subgroups of cells are shown in Table 3. Increase in T-cell population and decrease in monocytes and B-cells was seen during the first 24 h of PHA-stimulation. There was no difference in the distribution of different subclass’s of cells between cryopreserved and fresh samples. After about 48 h incubation, partly broken cells exciting light were seen in the microscope. The persistence of dead cells carrying surface markers displaying fluorescent light might have influenced the results, while cell count might include both living and dead cells.
4. Discussion Cryopreservation of blood samples for later analysis is a convenient method in biomonitoring studies but individual fresh and cryopreserved samples need to be compared to evaluate consequences of storing samples. Our study shows that fresh blood samples have lower DMS-induced DNA repair and background UDS compared to cryopreserved samples. The higher DNA repair found in cryopreserved samples might be due to a selection or damage of cells during freezing and thawing of samples. Cells with a low DNA repair activity or otherwise vulnerable might be lost, leading to a higher proportion of cells with a high repair activity. Higher UDS in frozen samples could also be the result of increased repair of damage due to higher sensitivity of cells. In order to minimise the cell loss, the thawing process was optimised to obtain a relative cell survival of more than 90% after removal of DMSO by centrifugation. Other studies have reported comparable cell survival w19x. Furthermore, we found that fresh blood samples have higher DNA repair activity in cells incubated with autologous plasma compared to FBS. When comparing values obtained by use of the two stimulation factors a significant correlation was found. This indicates a systematically higher level of UDS in autologous incubation media. Previous reports
also found highest values with autologous plasma in incubation media possibly due to higher extent of stimulation compared with FBS w20x. There was no difference found in the DNA repair activity of cryopreserved cells when either autologous plasma or FBS were included. Two parameters might have influenced this; the storing of autologous plasma might somehow decrease the content of stimulating factors or a selection of cells during freezing and thawing may decrease the influence of plasmarserum used. The use of autologous plasma in biomonitoring studies may however cause problems when samples are stored as we found it very unstable when frozen, due to partial coagulation Žunpublished observations.. Thus, fresh human plasma Žnot necessarily autologous. is recommended. Reported CE Žmean 31%r30%. and MF Žmean 13.7r10.0 = 10y6 . in this study of fresh and cryopreserved samples are within the range of what has been reported previously w13x. The mean MF in comparable samples from individuals analysed in different laboratories has been found to vary by at least one order of magnitude Ž1.1–32.9 = 10y6 .. This variation is partly related to the differences in age, gender and smoking habits of the donorsrsubjects studied. In the present study, the results of the individual mutant frequency ŽMF. in cryopreserved samples are significantly lower than in fresh samples. However, the Pearson statistical correlation analysis revealed an association between individual fresh and frozen samples. This finding justifies the use of cryopreserved material in MF analysis where storage is necessary. The lower MF in cryopreserved samples may be due to a difference in cell ‘quality’ after the freezingrthawing process. The content of T cells present in individual samples before PHA stimulation was found to positively influence the CE in fresh samples. This is fully consistent with the assay being a T cell cloning assay, with phytohemaglutinin as a primarily T-cell stimulation agent. However, our results show a minor proportion of B-cells and monocytes to persist in media. Analysis of the contribution of subcell concentration measured immediately before incubation Ž n s 8. t s 0, showed significant positive effect of the content of T cells and monocytes in fresh samples on CE, while no significant contribution was found on the MF. In frozen samples Ž n s 8. no significant
L. Risom, L.E. Knudsenr Mutation Research 440 (1999) 131–138
influence of subcell concentration was found neither in CE nor MF. The impact of storing samples for later analysis has been investigated in other studies. DNA damage monitored by the Comet assay showed no differences in the use of cryopreserved cells vs. fresh cells in exposure assessment w21x. Cryopreserved material has also been proven comparable to fresh material in the T cell cloning assay w22x. In a study of chromosomal aberrations ŽCA. a significant difference was found between cryopreserved samples and fresh samples in the number of CA w23x. A higher number of CA was found in cryopreserved samples and is suggested to be associated with unidentified factors in the freezingrthawing or storage procedure. However, a recent study on chromosomal aberrations did not report any significant effect of cryopreservation of lymphocytes w24x. No measure of the relative cell survival is given after the freezingrthawing process. Another study has found an intact capacity of lymphokine production in T cells after cryopreservation assuming this as an indication of a good maintenance of T cell function w19x. Our study shows that results obtained by use of cryopreserved samples are not directly comparable with fresh samples. However, a positive association was found between fresh and cryopreserved samples in both assays. Furthermore, we recommend only fresh Žand not frozen. human plasma to be used in UDS measurements.
Acknowledgements We are thankful to staff members of the Department of Chemical Working Environments, National Institute of Occupational Health. The financial support of the Danish Cancer Society in form of scholarship to LR is gratefully acknowledged.
References w1x P. Møller, L.E. Knudsen, G. Frenz, M. Dybdahl, H. Wallin, B.A. Nexø, Seasonal variation of DNA damage and repair in patients with non-melanoma skin cancer and referents with and without psoriasis, Mutation Res. 407 Ž1998. 25–34.
137
w2x P.B. Farmer, O. Sepai, R. Lawrence, H. Autrup, P.S. Nielsen, A.B. Vestergard, ˚ R. Waters, C. Leuratti, N.J. Jones, J. Stone, R.A. Baan, J.H.M. van Delft, M.J.S.T. Steenwinkel, S.A. Kyrtopoulos, V.L. Souliotis, N. Theodorakopoulos, N.C. Ba˚ Ancalis, A.T. Natarajan, A.D. Tates, A. Haugen, A. dreassen, S. Øvrebø, D.E.G. Shuker, K.S. Amaning, A. Schouft, A. Ellul, R.C. Garner, K.H. Dingley, A. Abbondandolo, F. Merlo, J. Cole, K. Aldrich, D. Beare, E. Capulas, G. Rowley, A.P.W. Waugh, A.C. Povey, K. Haque, M. KirschVolders, P. van Hummelen, P. Castelain, Biomonitoring human exposure to environmental carcinogenic chemicals, Mutagenesis 11 Ž1996. 363–381. w3x M.H.L. Green, C.F. Arlett, G. Bentham, G. Cortopassi, Y. Liu, H. Steingrimsdottır, ¨ E. Capulas, J. Cole, Is there a link between sunlight and non-hodgkin lymphoma ŽNHL.?, Mutation Res. 379 Ž1997. 187, Suppl. 1. w4x L.E. Knudsen, L.P. Ryder, K. Wassermann, Induction of DNA repair synthesis in human monocytesrB-lymphocytes compared with T-lymphocytes after exposure to N-acetyl-Nacetylaminofluorene and dimethylsulfate in vitro, Carcinogenesis 13 Ž1992. 1285–1287. w5x K. Savela, K. Hemminki, DNA adducts in lymphocytes and granulocytes of smokers and nonsmokers detected by the 32P-postlabelling assay, Carcinogensis 12 Ž1991. 503–508. w6x R. Benigni, A. Calcagnile, G. Fabri, A. Giuliani, P. Leopardi, A. Paoletti, Biological monitoring of workers in the rubber industry: II. UV-induced unscheduled DNA synthesis in the lymphocytes of vulcanizers, Mutation Res. 138 Ž1984. 105– 111. w7x L.E. Knudsen, T. Boisen, J.M. Christensen, J.E. Jelnes, G.E. Jensen, J.C. Jensen, K. Lundgren, C. Lundsteen, B. Pedersen, K. Wassermann, P. Wilhardt, H.C. Wulf, U. Zebitz, Biomonitoring of genotoxic exposure among stainless steel welders, Mutation Res. 279 Ž1992. 129–143. w8x S.-M. Hou, B. Lambert, K. Hemminki, Relationship between hprt mutant frequency, aromatic DNA adducts and genotypes for GSTM1 and NAT2 in bus maintenance workers, Carcinogenesis 16 Ž1995. 1913–1917. w9x J. Cole, M.H.L. Green, S.E. James, L. Henderson, H. Cole, A further assessment of factors influencing measurements of thioguanine-resistant mutant frequency in circulating Tlymphocytes, Mutation Res. 204 Ž1988. 493–507. w10x P. Vodicka, T. Bastlova, L. Vodickova, K. Peterkova, B. Lambert, K. Hemminki, Biomarkers of styrene exposure in lamination workers: levels of O 6-guanine DNA adducts, DNA strand breaks and mutant frequencies in hypoxanthine guanine phosphoribosyltransferase gene in T-lymphocytes, Carcinogenesis 16 Ž1995. 1473–1481. w11x P.S. Nielsen, H. Okkels, T. Sigsgaard, S. Kyrtopoulos, H. Autrup, Exposure to urban and rural air pollution: DNA and protein adducts and effect of glutathione-S-transferase genotype on adduct levels, Int. Arch. Occ. Environ. Health 68 Ž1996. 170–176. w12x M. Dybdahl, G. Frentz, U. Vogel, H. Wallin, B.A. Nexø, Low DNA repair is a risk factor in skin carcinogenesis. A study of basal cell carcinoma in psoriasis patients, Mutation Res., in press.
138
L. Risom, L.E. Knudsenr Mutation Research 440 (1999) 131–138
w13x J. Cole, T.R. Skopek, Somatic mutant frequency, mutation rates and mutational spectra in the human population in vivo, Mutation Res. 304 Ž1994. 33–105. w14x L.S. Olsen, L.R. Nielsen, B.A. Nexø, K. Wassermann, Somatic mutation detection in human biomonitoring, Pharmacology and Toxicology 78 Ž1996. 364–373. w15x L.E. Knudsen, Evaluation of unscheduled DNA synthesis ŽUDS. in human lymphocytes as an indicator of genotox exposure. Use and validation of a selected method for monitoring and screening for occupational genotoxic exposure, PhD Thesis, National Institute of Occupational Health, Copenhagen, 1993, pp. 1–85. w16x R.W. Pero, M. Lund-Pero, The reproducibility of individual estimations in the covalent binding of N-acetoxy-2acetylaminofluorene to DNA and in the induction of unscheduled DNA synthesis, Mutation Res. 120 Ž1983. 219– 224. w17x R.J. Albertini, K.L. Castle, W.R. Borcherding, T-cell cloning to detect the mutant 6-thioguanine-resistant lymphocytes present in human peripheral blood, Proc. Natl. Acad. Sci. U.S.A. 79 Ž1982. 6617–6621. w18x S.-M. Hou, S. Falt, ¨ A. Steen, Hprt mutant frequency and GSTM1 genotype in non-smoking healthy individuals, Environ. Mol. Mutagen. 25 Ž1995. 97–105.
w19x S.-Y. Wang, M.-L. Hsu, C.-H. Tzeng, H.-C. Hsu, C.-K. Ho, The influence of cryopreservation on cytokine production by human T lymphocytes, Cryobiology 37 Ž1998. 22–29. w20x L.L. Larcom, M.E. Smith, Quantitative assay for evaluating immunocompetence and DNA-repair capacity, J. Natl. Cancer Inst. 80 Ž1988. 1112–1118. w21x E.-E. Visvardis, A.M. Tassiou, S.M. Piperakis, Study of DNA damage induction and repair capacity of fresh and cryopreserved lymphocytes exposed to H 2 O 2 and gamma irradiation with the alkaline comet assay, Mutation Res. 383 Ž1997. 71–80. w22x J.P. O’Neill, J.A. Nicklas, T.C. Hunter, O.B. Batson, M. Allegretta, M.T. Falta, R.F. Branda, R.J. Albertini, The effect of T-lymphocyte ‘clonality’ on the calculated hprt mutation frequency occurring in vivo in humans, Mutation Res. 313 Ž1994. 215–225. w23x L.G. Littlefield, E.E. Joiner, S.P. Colyer, E.L. Frome, Effects of cryopreservation on the recovery of radiation-induced chromosome aberrations in human lymphocytes, Radiation Protection Dosimetry 18 Ž1986. 25–29. w24x V. Chambrette, P. Laroche, J.J. Lataillades, P. Voisin, Effect of cryopreservation on chromosomal aberration yield in irradiated lymphocytes, Int. J. Radiat. Biol. 70 Ž1996. 671–675.
Use of cryopreserved peripheral mononuclear blood cells in biomonitoring Lotte Risom b
a,)
, Lisbeth E. Knudsen
b
a Danish Cancer Society, StandbouleÕarden 49, DK-2100 Copenhagen Ø, Denmark National Institute of Occupational Health, Lersø Park Alle´ 105, DK-2100 Copenhagen Ø, Denmark
Received 2 October 1998; received in revised form 2 February 1999; accepted 4 February 1999
Abstract This study was performed to investigate the effect of storing blood samples by freezing on selected biomarkers and possible implications for biomonitoring. Comparative measurements were performed in order to investigate the use of cryopreserved vs. freshly separated peripheral mononuclear blood cells ŽPMBC. obtained from donor blood. Measurements of DNA-repair, mutant frequency, and subcell content were included. Samples for large biomonitoring studies are usually taken from study groups within a short timeperiod of daysrweeks and storing of study material for later analysis can be necessary. We measured the DNA repair activity as dimethylsulfate induced unscheduled DNA synthesis ŽUDS. in PMBC incubated with either autologous plasma or fetal bovine serum ŽFBS.. Comparison of the hprt mutant frequency by the T cell cloning assay was made in parallel. Finally the content of BrT-lymphocytes and monocytes was measured in phytohemaglutinin ŽPHA.-stimulated cultures at different time intervals. The results showed a higher DNA repair activity in cryopreserved samples compared with fresh samples. We also found differences in mutant frequencies with higher values in fresh samples. A significant correlation of frequencies was seen when comparing fresh with cryopreserved samples. Furthermore we recommend fresh human plasma used in UDS incubation media. q 1999 Elsevier Science B.V. All rights reserved. Keywords: Biomonitoring; Peripheral mononuclear blood cell; Cryopreservation; Storing
1. Introduction In large biomonitoring studies sampling logistics often puts demands for the storing of sample material for later analysis. First of all it is usually not possible to analyse all the samples within the study group on the day of sampling, because of the hours spent during the ) Corresponding author. Fax: q45-35-25-77-31; E-mail: [email protected]
collection of materials and the processing of samples and assaying due to time-consuming methods. Secondly seasonal variation of individual DNA damage and DNA repair has been reported. During summertime the sunlight exposure is high because of sunnier weather, increased outdoor life and less covering of the skin. Also changes in lifestyle, e.g., food consumption may account for seasonal variations. An association in the level of DNA damage and DNA repair with season in Denmark has indicated an association with sunlight exposure w1x. Also a
1383-5718r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved. PII: S 1 3 8 3 - 5 7 1 8 Ž 9 9 . 0 0 0 1 9 - 4
132
L. Risom, L.E. Knudsenr Mutation Research 440 (1999) 131–138
seasonal variation in measurements of micronuclei and sister chromatid exchange with higher levels in summertime has been reported w2x. Another study found a positive association between hprt mutant frequency in peripheral mononuclear blood cells ŽPMBC. and sunlight records in the United Kingdom w3x. The influence of seasonal variation on the sample material should be taken into account when comparing data. To minimise any seasonal variation between study persons, samples should therefore preferably be collected during a short period of time. Freezing blood cells may however introduce changes in the content of different subpopulations of white blood cells, due to heterogeneity in survival in the cell population as a result of the freezingrthawing process of cryopreserved samples. Changes in the proportion of subcells after storage could contribute to differences in, e.g., DNA repair activity and mutant frequency. Studies have reported a higher DNA repair activity in monocytes and B-lymphocytes compared to T-lymphocytes w4x. Other studies have revealed heterogeneity, e.g., a higher level of adducts in smokers’ lymphocyte DNA compared with granulocyte DNA w5x. Furthermore, freezing andror thawing may introduce DNA damage or mutations from endogenous processes in the cell. Therefore, one may not be able to compare results from fresh and cryopreserved samples. With the need in the nearby future, for storing large amounts of samples in biomonitoring studies for later analysis, we have investigated the impact of freezing blood samples. To evaluate the effect of cryopreservation we examined the DNA repair activity measured by dimethylsulfate ŽDMS. induced unscheduled DNA synthesis ŽUDS.. The T cell cloning assay was used in measurements of mutations in fresh and frozen PMBCs. Changes in cell population were determined in subpopulations of blood cells at different times of growing. UDS and the T cell cloning are both assays used in biomonitoring as measurements of individual environmental and occupational exposure. Measure of UDS has been used in studies of vulcanizers, stainless steel welders and sun exposure w6,7,1x. UDS is considered an indicator of cancer susceptibility following the important role of DNA repair in carcinogenesis. A low DNA repair is associated with higher cancer risk. Several studies have measured mutational effect in the hprt gene in
relation to exposure to diesel exhaust in bus drivers, to smoking, and to styrene in lamination workers w8–10x. Cryopreservation of PMBC samples for later analysis is a common and convenient procedure in biomonitoring studies, with measurements of, e.g., 32 P-postlabelling, host cell reactivation assay and T cell cloning w11–13x, while no studies of UDS have been found measured in cryopreserved samples.
2. Materials and methods 2.1. Cell biology All cell culture media, fetal bovine serum ŽFBS. and antibiotics were from Gibco BRL. The blood banks at Bispebjerg Hospital and Rigshospitalet, Copenhagen kindly supplied buffy coats and human serum ŽHS.. Dimethylsulfoxide ŽDMSO. and dimethylsulfate ŽDMS. were from Merck and Fluka respectively. Hydroxyurea ŽHU., phytohemaglutinin ŽPHA. and 6-thioguanine ŽTG. were from Sigma. g-Irradiation of lymphocytes was carried out at Department of Clinical Immunology, Rigshospitalet, Copenhagen. 2.2. Isolation of peripheral mononuclear blood cells Buffy coats from healthy donors were obtained from approximately 500-ml venous blood. Isolation of the peripheral mononuclear blood cells was done by gradient centrifugation 30 min at 800 = g using Lymphoprep w ŽNycomed Pharma AS, r s 1.077.. The autologous plasma was recovered from the upper phase of the separated cell layer. The mononuclear cells were isolated from the mid-layer and washed twice in cold 1 = PBS before suspension in basic medium ŽBM.. 2.3. Culture media BM is RPMI 1640 supplemented with 25 mM hepes, 100 IUrml benzylpenicilium and 100 mgrml streptomycin. Nutrient Medium ŽNM. is BM with 5% heat inactivated FBS and 5% HS. Growth medium ŽGM. is NM with 20% AIM V Žserum-free lymphocyte medium used for culturing functionally
L. Risom, L.E. Knudsenr Mutation Research 440 (1999) 131–138
differentiating lymphoid cell lines., 1 mgrml PHA and 20% T-cell growth factor enriched Conditioned Medium ŽCM. w14x. Both CM and HS were tested for the ability to promote long-term proliferation of activated T-cells in microplates and stored at y208C. 2.4. Freezing and thawing of cells Cells for freezing were adjusted to 1 = 10 7 cellsrml in cold BM containing 50% FBS and 10% DMSO. Immediately after suspension in media cells were transferred to cold cryotubes, stored in isopropanol containers at y708C Ž18Crmin. for 24 h before being transferred for storing at liquid nitrogen. Thawing of cells was done fast by incubation at 378C in water bath, gently turning the tubes a few times. When 9r10 of the cell suspension was thawed it was slowly mixed with 9 ml cold media containing 10% wrw dextrose ŽDifco, Detroit MI, USA., 40% RPMI 1640 and 50% FBS, before centrifugation at 800 = g for 10 min. The cells were kept on ice bath prior to analysis. 2.5. Unscheduled DNA synthesis The UDS protocol was modified from w15,16x. UDS is an indirect measure of overall DNA repair of induced damage. The isolated peripheral mononuclear blood cells were counted in haemacytometer and adjusted to 2 = 10 6 cellsrml in BM. For in vitro damage 2 = 10 6 cells, were incubated with 0 or 10 mM DMS for 30 min at 378C. After centrifugation DMS was removed and the cells resuspended in BM. The UDS was measured by incorporation of w3 Hxthymidine into DNA. 2 = 10 5 cells were dissolved in BM with 20% autologous plasma, 20% HS or 20% FBS and incorporated with 0.4 mM w3 Hxthymidine Ž25 Cirmmol, Amersham. at 378C for 18 h in microtiter wells Ž96 wells, 7 mm round bottom, Nunc.. Background UDS was determined in parallel cultures exposed to DMSO. To minimise replication of DNA, the cells were incubated with 10 mM of HU. The measurements were performed in replicates of eight in 96 microtiter wells. The cells were harvested in a cell harvester ŽMach 96, Wallac. transferring the washed cells onto a glass fibre filter, which was counted in a betaplate scintillation counter. The UDS results are given as cpm w3 Hxthymidine
133
incorporated into 2 = 10 5 cells, exposed to DMS, background subtracted. 2.6. T cell cloning The T cell cloning assay was performed as described by w17x with modifications made by w14,18x. After washing twice in cold PBS lymphocytes were resuspended in cold NM at 1 = 10 6 cellsrml. Cells were then incubated at 378C with 44 mgrml PHA for 44 h. Two non-selective plates were prepared by inoculating 2 target cells and 2 = 10 4 freshly g-irradiated Ž40 Gy. lymphoblastoid RJK853 cells Žkindly provided by Dr. Hou at Environmental Medicine Unit, Karolinska Institute, CNTrNOVUM, Huddinge, Sweden. as feeder cells in 200 ml GM in each well. Selection plates received 2 = 10 4 target cells and 1 = 10 4 feeder cells per well containing 2 mgrml TG Ž12 mM.. A stock solution of 1 mgrml of TG in 0.01 M NaOH was made immediately before use. The plates were wrapped in plastic foil to avoid evaporation and incubated at 378C in 5% CO 2 in air at 95% humidity for 2 weeks without medium change. Wells were then scored visually for the presence of clones in the wells using an inverted microscope. After 3–4 days the plates were inspected again to check whether slowly growing clones had been missed. Cloning efficiency ŽCE. with and without TG was calculated from the proportion of negative wells Ž P0 . assuming a Poisson distribution of clone forming cells between wells, i.e., P0 s eyx , and CE s xrn, where n s number of cells seeded per well. The frequency of mutant cells ŽMF. was given by CE in the presence of TG divided by the CE in the absence of TG w17x. 2.7. Quantificationr distribution of PMBC content Quantification of content of B- and T-lymphocytes and monocytes in peripheral mononuclear blood cell fractions was measured by incubation with PHA for 0, 24, 48, 72 and 96 h. Samples of PMBC was incubated with specific monoclonal antibodies towards T-lymphocytes ŽAnti-Leu-4-CD3. and Blymphocytes Ž Anti-Leu-12-CD19 . Ž Simultest CD3rCD19, Becton Dickinson.. A separate cell sample was incubated with antibodies towards
L. Risom, L.E. Knudsenr Mutation Research 440 (1999) 131–138
134
monocytes ŽAnti-Leu-M3-CD14, PE Becton Dickinson.. The monoclonal antibodies are conjugated to different fluorochromes: fluorescein conjugate or phycoerythrin conjugate. When antibodies bound, cells are excited by a single light source from a fluorescence microscope, T-cells display green fluorescence Žfluorescin. and B-cells orange-red fluorescence Žphycoerythrin.. 5 = 10 5 cells were incubated with 10 ml antibodies for 30 min on ice in a total volume of 20 ml. Slides were prepared using 10 ml of the cell suspension. The slide was covered with coverslip, protected from light and allowed to stand for 2 min. The contents of green and orange fluorescing cells and the total number of cells were counted at 40 = in the epi-fluorescence microscope. To count the total amount of cells the microscope was switched from epi-fluorescence to transmission of visible light. The number of B- and T-cells and the total number of cells were counted Žminimum 100 cells.. In the separate sample staining monocytes the number of monocytes and the total number of cells were counted. The subcell populations are calculated as percentage of cells in the sample.
2.8. Statistics The means, standard deviations and correlations were calculated by use of the SAS computer program. The UDS measurements were compared by analysis for significant differences among mean values by the non-parametric Mann–Whitney statistical analysis. The Student’s t-test was used to compare the cloningrmutant frequency of cryopreserved vs. fresh samples. The Pearson correlation analysis was performed to test for systematic differences, e.g., higher levels in fresh compared with cryopreserved samples. The level of significance was 5%.
3. Results 3.1. UDS in primary human PMBCs A total of 19 healthy persons were used to measure background UDS and DNA repair activity of DMS induced UDS minus background UDS of PMBCs obtained from buffy coats ŽTable 1.. Due to
Table 1 DNA repair in fresh and cryopreserved PMBCs using autologous plasma in incubation media Donor code
Fresh samples
Cryopreserved samples
DMSO Žbackground.
DMS Ž10 mM. induced UDS
DNA repair ŽDMS–DMSO.
DMSO Žbackground.
DMS Ž10 mM. induced UDS
a DNA repair ŽDMS–DMSO.
39 40 71 06 50 98 58 59 940 941 942 960 961 962
137b 126 182 107 68 107 191 156 256 266 242 108 133 174
288 252 155 193 92 174 205 231 257 295 313 136 106 239
151 126 y27 86 24 67 14 75 1 29 71 28 y27 65
136 238 169 132 213 322
325 607 255 333 321 576
189 369 86 201 108 254
c
c
c
c
c
c
142 198 153 202 259 171
198 331 291 270 346 216
56 133 138 68 87 45
Mean " S.D.
161 " 61
210 " 70
49 " 53
195 " 57
339 " 127
145 " 95
a
a
DNA repair activity is DMS induced UDS minus DMSO background. Result given as cpm w3 Hxthymidine incorporated into 2 = 10 5 cells. c Samples where only autologous plasma for fresh samples could be collected. b
L. Risom, L.E. Knudsenr Mutation Research 440 (1999) 131–138 Table 2 Cloning efficiency and mutant frequency of 22 individual fresh and cryopreserved donor samples Donor code
Fresh samples
Cryopreserved samples y6
CEr%
MF=10
CEr%
MF=10y6
54 59 61 65 85 92 39 40 31 35 71 04 83 84 88 98 63 64 78 80 064 088
28 19 30 37 34 24 44 40 16 36 49 44 10 22 36 37 37 47 28 31 12 12
10.3 4.1 15.8 11.8 11.2 30.3 8.6 4.7 32.0 6.1 5.2 12.7 19.5 8.2 8.8 5.4 6.4 7.9 6.6 9.2 39.7 36.7
21 39 40 23 37 24 49 50 33 21 26 37 31 26 25 18 28 27 20 35 26 24
7.6 12.9 13.3 9.3 3.3 39.0 4.2 1.7 7.8 4.3 1.1 7.8 7.2 10.3 10.7 12.1 5.7 12.2 9.6 6.3 18.5 17.1
Mean"S.D.
31"12
13.7"10.9
30"9
10.0"7.7
135
incubated with autologous plasma showed significantly higher values in cryopreserved samples p s 0.004, n s 12 ŽTable 1.. Additionally, samples with FBS in incubation media showed significantly higher values in cryopreserved samples p s 0.002, n s 12 Ždata not shown.. In fresh samples significantly higher UDS values were found in samples incubated with autologous plasma compared with samples incubated with FBS p s 0.01, n s 14 Žwhereas in frozen samples p s 0.22, n s 8 when comparing incubation media.. Values obtained by use of autologous plasma were tested for correlation with values of FBS in incubation medium. A significant correlation was found with r s 0.58, p s 0.03, n s 14 and r s 0.92, p s 0.001, n s 8 for fresh and cryopreserved samples, respectively.
3.2. Mutant frequency in PMBCs
CE, cloning efficiency; MF, mutation frequency; S.D., standard deviation.
lack of induced repair some samples exhibited negative values. FBS was tested as enrichment in medium as substitute for autologous plasma, which was only available for 14 fresh samples and 12 cryopreserved samples. The results of background and DNA repair activity UDS in fresh and cryopreserved samples
A total of 22 donor samples could be used in the statistical calculations of both CE and MF between pair measurements of individual fresh and cryopreserved samples ŽTable 2.. MF in fresh samples yielded mean of 13.7 " 10.9 Ž=10y6 . and cryopreserved samples yielded mean MF of 10.0 " 7.7 Ž=10y6 .. The Student’s t-test revealed significant difference Ž p s 0.001.. To test for association between fresh and cryopreserved samples of individual MF measurements the Pearson correlation was calculated and found significant Ž r s 0.58, p s 0.004, n s 22., which implies association. Fresh samples yielded mean CE of 31% " 12 and cryopreserved samples yielded mean CE of 30% " 9 after cloning. No difference was seen Ž p s 0.82..
Table 3 Variation in concentrations of monocytes, B-and T-cells in PHA stimulated PMBCs at different times during incubation PHArhour a
0 24 48 72 96 a
N
8 6 8 8 6
Freshly isolated PMBC
N
%B
%T
%Mono
12 8 10 8 6
49 64 56 70 86
34 26 20 21 11
6 3 6 6 4
Cryopreserved PMBC %B
%T
%Mono
12 8 5 9 7
70 86 84 79 79
27 10 15 16 16
Phytohemaglutinin ŽPHA. stimulation per hour. N, Number of samples; PMBC, peripheral mononuclear blood cell; B, B-cells; T, T-cells; Mono, monocytes.
136
L. Risom, L.E. Knudsenr Mutation Research 440 (1999) 131–138
3.3. Variation in subgroups of PMBCs The content of monocytes, T- and B-cells were measured just before incubation Ž t s 0. and after 24 Ž t s 24., 48 Ž t s 48., 72 Ž t s 72. and 96 Ž t s 96. h of PHA-incubation. Measurements of variation in subgroups of cells are shown in Table 3. Increase in T-cell population and decrease in monocytes and B-cells was seen during the first 24 h of PHA-stimulation. There was no difference in the distribution of different subclass’s of cells between cryopreserved and fresh samples. After about 48 h incubation, partly broken cells exciting light were seen in the microscope. The persistence of dead cells carrying surface markers displaying fluorescent light might have influenced the results, while cell count might include both living and dead cells.
4. Discussion Cryopreservation of blood samples for later analysis is a convenient method in biomonitoring studies but individual fresh and cryopreserved samples need to be compared to evaluate consequences of storing samples. Our study shows that fresh blood samples have lower DMS-induced DNA repair and background UDS compared to cryopreserved samples. The higher DNA repair found in cryopreserved samples might be due to a selection or damage of cells during freezing and thawing of samples. Cells with a low DNA repair activity or otherwise vulnerable might be lost, leading to a higher proportion of cells with a high repair activity. Higher UDS in frozen samples could also be the result of increased repair of damage due to higher sensitivity of cells. In order to minimise the cell loss, the thawing process was optimised to obtain a relative cell survival of more than 90% after removal of DMSO by centrifugation. Other studies have reported comparable cell survival w19x. Furthermore, we found that fresh blood samples have higher DNA repair activity in cells incubated with autologous plasma compared to FBS. When comparing values obtained by use of the two stimulation factors a significant correlation was found. This indicates a systematically higher level of UDS in autologous incubation media. Previous reports
also found highest values with autologous plasma in incubation media possibly due to higher extent of stimulation compared with FBS w20x. There was no difference found in the DNA repair activity of cryopreserved cells when either autologous plasma or FBS were included. Two parameters might have influenced this; the storing of autologous plasma might somehow decrease the content of stimulating factors or a selection of cells during freezing and thawing may decrease the influence of plasmarserum used. The use of autologous plasma in biomonitoring studies may however cause problems when samples are stored as we found it very unstable when frozen, due to partial coagulation Žunpublished observations.. Thus, fresh human plasma Žnot necessarily autologous. is recommended. Reported CE Žmean 31%r30%. and MF Žmean 13.7r10.0 = 10y6 . in this study of fresh and cryopreserved samples are within the range of what has been reported previously w13x. The mean MF in comparable samples from individuals analysed in different laboratories has been found to vary by at least one order of magnitude Ž1.1–32.9 = 10y6 .. This variation is partly related to the differences in age, gender and smoking habits of the donorsrsubjects studied. In the present study, the results of the individual mutant frequency ŽMF. in cryopreserved samples are significantly lower than in fresh samples. However, the Pearson statistical correlation analysis revealed an association between individual fresh and frozen samples. This finding justifies the use of cryopreserved material in MF analysis where storage is necessary. The lower MF in cryopreserved samples may be due to a difference in cell ‘quality’ after the freezingrthawing process. The content of T cells present in individual samples before PHA stimulation was found to positively influence the CE in fresh samples. This is fully consistent with the assay being a T cell cloning assay, with phytohemaglutinin as a primarily T-cell stimulation agent. However, our results show a minor proportion of B-cells and monocytes to persist in media. Analysis of the contribution of subcell concentration measured immediately before incubation Ž n s 8. t s 0, showed significant positive effect of the content of T cells and monocytes in fresh samples on CE, while no significant contribution was found on the MF. In frozen samples Ž n s 8. no significant
L. Risom, L.E. Knudsenr Mutation Research 440 (1999) 131–138
influence of subcell concentration was found neither in CE nor MF. The impact of storing samples for later analysis has been investigated in other studies. DNA damage monitored by the Comet assay showed no differences in the use of cryopreserved cells vs. fresh cells in exposure assessment w21x. Cryopreserved material has also been proven comparable to fresh material in the T cell cloning assay w22x. In a study of chromosomal aberrations ŽCA. a significant difference was found between cryopreserved samples and fresh samples in the number of CA w23x. A higher number of CA was found in cryopreserved samples and is suggested to be associated with unidentified factors in the freezingrthawing or storage procedure. However, a recent study on chromosomal aberrations did not report any significant effect of cryopreservation of lymphocytes w24x. No measure of the relative cell survival is given after the freezingrthawing process. Another study has found an intact capacity of lymphokine production in T cells after cryopreservation assuming this as an indication of a good maintenance of T cell function w19x. Our study shows that results obtained by use of cryopreserved samples are not directly comparable with fresh samples. However, a positive association was found between fresh and cryopreserved samples in both assays. Furthermore, we recommend only fresh Žand not frozen. human plasma to be used in UDS measurements.
Acknowledgements We are thankful to staff members of the Department of Chemical Working Environments, National Institute of Occupational Health. The financial support of the Danish Cancer Society in form of scholarship to LR is gratefully acknowledged.
References w1x P. Møller, L.E. Knudsen, G. Frenz, M. Dybdahl, H. Wallin, B.A. Nexø, Seasonal variation of DNA damage and repair in patients with non-melanoma skin cancer and referents with and without psoriasis, Mutation Res. 407 Ž1998. 25–34.
137
w2x P.B. Farmer, O. Sepai, R. Lawrence, H. Autrup, P.S. Nielsen, A.B. Vestergard, ˚ R. Waters, C. Leuratti, N.J. Jones, J. Stone, R.A. Baan, J.H.M. van Delft, M.J.S.T. Steenwinkel, S.A. Kyrtopoulos, V.L. Souliotis, N. Theodorakopoulos, N.C. Ba˚ Ancalis, A.T. Natarajan, A.D. Tates, A. Haugen, A. dreassen, S. Øvrebø, D.E.G. Shuker, K.S. Amaning, A. Schouft, A. Ellul, R.C. Garner, K.H. Dingley, A. Abbondandolo, F. Merlo, J. Cole, K. Aldrich, D. Beare, E. Capulas, G. Rowley, A.P.W. Waugh, A.C. Povey, K. Haque, M. KirschVolders, P. van Hummelen, P. Castelain, Biomonitoring human exposure to environmental carcinogenic chemicals, Mutagenesis 11 Ž1996. 363–381. w3x M.H.L. Green, C.F. Arlett, G. Bentham, G. Cortopassi, Y. Liu, H. Steingrimsdottır, ¨ E. Capulas, J. Cole, Is there a link between sunlight and non-hodgkin lymphoma ŽNHL.?, Mutation Res. 379 Ž1997. 187, Suppl. 1. w4x L.E. Knudsen, L.P. Ryder, K. Wassermann, Induction of DNA repair synthesis in human monocytesrB-lymphocytes compared with T-lymphocytes after exposure to N-acetyl-Nacetylaminofluorene and dimethylsulfate in vitro, Carcinogenesis 13 Ž1992. 1285–1287. w5x K. Savela, K. Hemminki, DNA adducts in lymphocytes and granulocytes of smokers and nonsmokers detected by the 32P-postlabelling assay, Carcinogensis 12 Ž1991. 503–508. w6x R. Benigni, A. Calcagnile, G. Fabri, A. Giuliani, P. Leopardi, A. Paoletti, Biological monitoring of workers in the rubber industry: II. UV-induced unscheduled DNA synthesis in the lymphocytes of vulcanizers, Mutation Res. 138 Ž1984. 105– 111. w7x L.E. Knudsen, T. Boisen, J.M. Christensen, J.E. Jelnes, G.E. Jensen, J.C. Jensen, K. Lundgren, C. Lundsteen, B. Pedersen, K. Wassermann, P. Wilhardt, H.C. Wulf, U. Zebitz, Biomonitoring of genotoxic exposure among stainless steel welders, Mutation Res. 279 Ž1992. 129–143. w8x S.-M. Hou, B. Lambert, K. Hemminki, Relationship between hprt mutant frequency, aromatic DNA adducts and genotypes for GSTM1 and NAT2 in bus maintenance workers, Carcinogenesis 16 Ž1995. 1913–1917. w9x J. Cole, M.H.L. Green, S.E. James, L. Henderson, H. Cole, A further assessment of factors influencing measurements of thioguanine-resistant mutant frequency in circulating Tlymphocytes, Mutation Res. 204 Ž1988. 493–507. w10x P. Vodicka, T. Bastlova, L. Vodickova, K. Peterkova, B. Lambert, K. Hemminki, Biomarkers of styrene exposure in lamination workers: levels of O 6-guanine DNA adducts, DNA strand breaks and mutant frequencies in hypoxanthine guanine phosphoribosyltransferase gene in T-lymphocytes, Carcinogenesis 16 Ž1995. 1473–1481. w11x P.S. Nielsen, H. Okkels, T. Sigsgaard, S. Kyrtopoulos, H. Autrup, Exposure to urban and rural air pollution: DNA and protein adducts and effect of glutathione-S-transferase genotype on adduct levels, Int. Arch. Occ. Environ. Health 68 Ž1996. 170–176. w12x M. Dybdahl, G. Frentz, U. Vogel, H. Wallin, B.A. Nexø, Low DNA repair is a risk factor in skin carcinogenesis. A study of basal cell carcinoma in psoriasis patients, Mutation Res., in press.
138
L. Risom, L.E. Knudsenr Mutation Research 440 (1999) 131–138
w13x J. Cole, T.R. Skopek, Somatic mutant frequency, mutation rates and mutational spectra in the human population in vivo, Mutation Res. 304 Ž1994. 33–105. w14x L.S. Olsen, L.R. Nielsen, B.A. Nexø, K. Wassermann, Somatic mutation detection in human biomonitoring, Pharmacology and Toxicology 78 Ž1996. 364–373. w15x L.E. Knudsen, Evaluation of unscheduled DNA synthesis ŽUDS. in human lymphocytes as an indicator of genotox exposure. Use and validation of a selected method for monitoring and screening for occupational genotoxic exposure, PhD Thesis, National Institute of Occupational Health, Copenhagen, 1993, pp. 1–85. w16x R.W. Pero, M. Lund-Pero, The reproducibility of individual estimations in the covalent binding of N-acetoxy-2acetylaminofluorene to DNA and in the induction of unscheduled DNA synthesis, Mutation Res. 120 Ž1983. 219– 224. w17x R.J. Albertini, K.L. Castle, W.R. Borcherding, T-cell cloning to detect the mutant 6-thioguanine-resistant lymphocytes present in human peripheral blood, Proc. Natl. Acad. Sci. U.S.A. 79 Ž1982. 6617–6621. w18x S.-M. Hou, S. Falt, ¨ A. Steen, Hprt mutant frequency and GSTM1 genotype in non-smoking healthy individuals, Environ. Mol. Mutagen. 25 Ž1995. 97–105.
w19x S.-Y. Wang, M.-L. Hsu, C.-H. Tzeng, H.-C. Hsu, C.-K. Ho, The influence of cryopreservation on cytokine production by human T lymphocytes, Cryobiology 37 Ž1998. 22–29. w20x L.L. Larcom, M.E. Smith, Quantitative assay for evaluating immunocompetence and DNA-repair capacity, J. Natl. Cancer Inst. 80 Ž1988. 1112–1118. w21x E.-E. Visvardis, A.M. Tassiou, S.M. Piperakis, Study of DNA damage induction and repair capacity of fresh and cryopreserved lymphocytes exposed to H 2 O 2 and gamma irradiation with the alkaline comet assay, Mutation Res. 383 Ž1997. 71–80. w22x J.P. O’Neill, J.A. Nicklas, T.C. Hunter, O.B. Batson, M. Allegretta, M.T. Falta, R.F. Branda, R.J. Albertini, The effect of T-lymphocyte ‘clonality’ on the calculated hprt mutation frequency occurring in vivo in humans, Mutation Res. 313 Ž1994. 215–225. w23x L.G. Littlefield, E.E. Joiner, S.P. Colyer, E.L. Frome, Effects of cryopreservation on the recovery of radiation-induced chromosome aberrations in human lymphocytes, Radiation Protection Dosimetry 18 Ž1986. 25–29. w24x V. Chambrette, P. Laroche, J.J. Lataillades, P. Voisin, Effect of cryopreservation on chromosomal aberration yield in irradiated lymphocytes, Int. J. Radiat. Biol. 70 Ž1996. 671–675.