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Laser scanning microscopic analysis of DNA damage in frozen tissues
CANCER LETTERS Cancer
Laser scanning
Letters
76 (1994) 127-132
microscopic analysis in frozen tissues
Daryl William Fairbairn, Richard Van Grigsby, Department
ofMicrobiology. (Received
Brigham
25 October
of DNA damage
Walter August0 Reyes, Kim Leslie O’Neill*
Young University.
1993; accepted
Provo. UT 84602. US.4
8 November
1993)
Abstract
DNA damage is central to research in many fields, especially cancer research and toxicology. The possible loss of DNA structural integrity during freezing or sustained maintenance at low temperatures may present difficulties in the interpretation of data accumulated in studies of tissues collected over a period of time and subsequently evaluated. Using laser scanning microscopic analysis of the recently developed single-cell gel (SCG) assay to measure DNA strand breaks in individual cells, we found that the basal levels of DNA damage in frozen tissue was higher than fresh tissue, but tissues frozen for greater lengths of time do not appear to contain significantly more DNA damage than those frozen for a short period. Evaluation of DNA damage in tumors stored by or collected using cryopreservation may produce artificially exaggerated levels of damage, which could limit analytical interpretations. Key words:
Single-cell
gel assay; DNA breaks:
Laser scanning
1. Introduction DNA
damage
is central
to research
in many
and toxicology. Assays that measure DNA damage are routinely used to determine the effects of chemicals on cells, the capacity of cells to repair induced DNA damage and the potentia response of tumors to chemotherapeutics and radiotherapeutics. Samfields,
especially
* Corresponding
cancer
research
author.
0 1994 Elsevier Scientific 0304~3835/94/$06.00 SSDI 0304-3835(93)03231-S
Publishers
Ireland
microscopy
ples of tissues collected for subsequent DNA analysis are routinely stored at low temperatures. When evaluating the data collected from experiments using such samples, the possibility of DNA degradation or some other alteration in the DNA of stored tissues that could change the profile of DNA damage interpretation is an area of concern. The single-cell gel assay [1 l] is a rapid and sensitive technique for the detection of DNA strand breaks in individual cells. It was originally designed to detect DNA double-strand breaks [lo]. Ltd. All rights reserved
128
Independent modifications of the technique provided a means of detecting DNA single-strand breaks under DNA denaturing conditions [4,14]. Applications for the single-cell gel assay are rapidly expanding into many fields. It was conceived for the determination of DNA damage inflicted with various types of radiation, and was used almost immediately to evaluate tumor responses to radiotherapy in a clinical environment [12]. It was also recognized to have excellent potential in identifying heterogeneous responses within cell populations due to specific subsets of cells. aiding in the identification and treatment of hypoxic cells [6]. which have proved to be significantly more resistant to radiotherapy, as well as the identification of cells resistant to chemotherapeutic agents such as etoposide [5,7,8]. Similar studies may become very useful in predicting tumor responses to various types of therapy on an individual basis (see Ref. 2 for a review of the single-cell gel assay). The effect of freezing and thawing has been examined in peripheral blood lymphocytes using DNA fingerprinting techniques [13] and spectrophotometric analysis of DNA fragments [3]. Both these reports were consistent with a recent report evaluating the effects of cryopreservation on peripheral blood lymphocytes using the SCG assay [l5]. These reports indicate that cryopreservation does not cause a significant increase in the levels of single or double-strand DNA breaks. M’e were concerned that cryopreservation may alter the basal levels of DNA strand breaks in frozen solid tissues. The purpose of this study was to determine the effects of freezing, including repeated freezing and thawing, as well as the effects of the length of storage at low temperatures, on the basal levels of DNA damage in individual cells from frozen tissue using the SCG assay. 2. Materials and methods 2.1. Tissues Male 2.5-week old BALBic mice were used in these experiments. Animals were sacrificed by cervical dislocation, and the spleen, brain, kidney, thymus and liver were immediately removed. Small pieces of tissue, approximately 20 mg each, were frozen to -70°C. Tissues were thawed in a
D. W. Fairhairn et al. /Cancer
Lett. 76 (19941 127-132
37°C water bath and refrozen (at least 1 h elapsed to allow complete freezing of the small pieces of tissue) up to twenty times. Unfrozen control tissues were used immediately upon extraction without freezing. Tissues remained frozen for the same length of time (3-4 days), except where indicated in the long-term freezing experiments (90 days). 2.2. Single-cell gel ussa?’ The SCG assay was performed essentially as described in a previous paper [9]. with some minor modifications. Approximately 20 mg of tissue was homogenized in 2-3 ml of PBS using a Yamoto LSC tissue homogenizer. Cells washed in PBS were suspended in 37°C low melting point agarose at a final concentration of 0.75”/;). A very thin gel was prepared on a fully frosted microscope slide. The slides were placed on ice to allow the agarose to gel and lysed in the dark in 1.0 M NaCl, 0.1% N-lauroyl sarcosine and 0.03 M NaOH for 60 min. The slides were washed in a solution of 0.03 M NaOH and 2 mM EDTA to remove the salt and detergents for an additional 60 min. and electrophoresed in a fresh solution for 25 min at 0.67 V/cm using a Biorad mode1 200/2.0 electrophoresis unit. They were then washed in distilled water and stained with 25 ~1 of 0.2 pgiml ethidium bromide for microscopic analysis. 2.3. Laser scanning microscopy and analysis A Carl Zeiss laser scanning microscope (Thornwood, NY) was used to analyze the comets in all the experiments, as previously described [l]. Cells were identified at 200x magnification and displayed on a Sony monitor by a scanning laser beam from a 5 mW argon laser (488/514 nm). Brightness and contrast were adjusted to clearly define the comet head and tail borders. Measurements were obtained by control of measuring cursors included as part of the microscope system software. Twenty-five comets from each slide were scored. Slides were scored in duplicate, and the experiments were repeated four times. 3. Results and discussion The unique
design
of the single-cell
gel assay
D. W. Fairbairn et al. /Cancer
Lett. 76 (1994)
127-132
30-
129
1+
20
U
frcsb kldaey frozen
kldncy
0 20
A.
40
60
Comet
20
30
C.
Length
fresh
l-+-
40
50
Comet
80
60
100
120
20
(microns)
llvcr
70
Length
B.
40
Comet
60
80
Length
100
120
(microns)
R
80
90
100
(microns)
0 20
D.
100
Co4Aet Le6nogth (ikrdns)
20U
fresh
tbymus
i G TO1
z
20
E. Fig. 1. Response
40
Comet
60
Length
80
100
120
(microns)
of tissue DNA damage
to freezing. (A) brain tissue; (B) kidney tissue; (C) liver tissue; (D) spleen tissue; (E) thymus tissue. Each sample represents 50 comets.
130
D. W. Fairbairn et al. /Cancer
Lett. 76 (1994)
127-132
100
2 i
4o
v
to
0
I brain
0
q
fresh 3 days
2
0
10 Number
kidney
liver
spleen
provides an excellent method for determining DNA strand breaks in individual cells. DNA suspended in agarose forms comets when subjected to an electric field. The comet migration pattern formed during electrophoresis is dependent upon the levels of DNA strand breaks in the cell. The degree of DNA damage is demonstrated in individual comets by the distance of DNA migration in the leading edge of the comet from the body of DNA, which is readily determined by subtracting the resulting comet lengths from unelectrophoresed control lengths. In order to evaluate the effects of freezing as a method of preservation of tissue samples for subsequent DNA damage analysis using the single-cell gel assay, we compared the DNA damage levels in frozen tissue samples with freshly obtained samples. Fig. 1 shows the distribution of comet lengths of both the fresh and frozen tissues for five tissue
freezing
and thawing
of
freeze/thaw
20 repeats
thymus
Fig. 2. Effect of freezingtime on DNA damage. Results of fresh samples are also shown for a comparison of 3-day with 90-day samples, Each bar represents the average of 50 comet lengths.
Table I Effect of repeated
I
60
on DNA migration
Fig. 3. Repeated freezing and thawing increases the levels of detectable basal DNA damage in tissues. Each point indicates the average of 50 comets.
types. There was a significant increase (P < 0.00 1) in basal DNA damage in all tissues examined. Note, however, that the increase in damage was different for each of the cell types. The increased migration ranged from only 11.6 pm for the liver sample to as much as 37.9 pm for the thymocytes. There was also a general increase in the heterogeneity of the comet distribution that occurred with freezing, as the range and standard deviations for the frozen samples were also greater (data not shown). Since we determined that there was an increase in detectable DNA damage in frozen tissues, we were interested in evaluating the effects of increased time spent at low temperatures on DNA basal damage. We compared samples of tissue that had been frozen for 3 days with samples that had been frozen for 90 days (Fig. 2). In all tissues except the liver there was an increase in the migration of
in tissue samples
Sample
Kidney Spleen Thymus
Fresh”
IX”
5x”
IOY”
20x”
7.1 +m 7.0 firn 12.4 pm
39.6 pm 34.7 pm 50.3 pm
44.7 pm 49.5 pm 61.1 pm
46.0 pm 53.8 pm 71.3 pm
50.2 pm 70.8 pm 70.1 pm
The averages of 50 comets are presented. “Distance of migration is determined by subtracting
the unelectrophoresed
control
length
from the comet length
D. W. Fairbairn et al. /Cancer
Lett. 76 (1994)
127-132
DNA with electrophoresis in the samples that had been frozen for the longer period. However, the increase was not statistically significant in the kidney (0.9 pm increase), spleen (0.8 pm increase) or thymus (2.8 pm increase). There was a significant (P c 0.01) increase in the migration (7.6 pm) of the sample frozen for a longer period only in the brain tissue. These results again demonstrate the heterogeneous effects of freezing, or the tissuespecific response to freezing, on the basal levels of DNA damage within the cells of given tissues. It does not appear that increased freezing time is correlaied with increased DNA damage in the individual cells of tissues. The third problem we examined was the effect of repeated freezing and thawing of tissues on DNA damage levels. Fig. 3 illustrates the results of our experiments. Freezing and thawing up to twenty times increased the comet length during electrophoresis by 10.6 pm in the kidney samples, 46.6 pm in the spleen and 20.0 ym in the thymus samples. We compared the lengths of unelectrophoresed control samples with repeated freezing and thawing in order to determine the distance of migration. The distance of migration was determined by subtracting the comet length from the length of the unelectrophoresed controls. There was some migration in the fresh samples, but the migration in the frozen samples was significantly greater. as previously mentioned (Table 1). The distance of migration increases with repetition of the freezing and thawing procedures (Table 1). The increased DNA migration due to this repetition was greatest in the spleen, where the migration distance increased from 24.2 pm in the samples frozen once to 70.8 pm in the samples frozen twenty times. As the single-cell gel assay continues to expand in application into new fields, more attempts will be made to evaluate the DNA damage levels and repair capacities of increasing cell and tissue types that have been exposed to a wide range of chemical, physical and physiological phenomena. Undoubtedly tissues and cells preserved for analysis will be evaluated. In fact such attempts have already been made. A paper evaluating the effects of high-dose combination alkylating agents with autologous bone-marrow support on the peri-
131
pheral lymphocytes of patients with breast cancer compared the effcts of cryopreservation of the lymphocytes on DNA damage levels, and found no increase in DNA migration due to freezing in these cells [ 151. The same group produced another paper later that examined radiation-induced damage. Again, cryopreserved peripheral lymphocytes were used [ 161. Our results indicate that there is a difference in the basal levels of DNA damage between fresh and frozen tissue samples. We found that of the five tissues evaluated. the greatest increase in detectable damage was demonstrated by the thymocytes (Fig. 1). The brain responded to increased length of time at low temperatures with the greatest increase in damage when compared with tissues frozen for a shorter period. None of the other tissues appeared to be affected by increasing the length of freezing time on expression of DNA damage (Fig. 2). Repeated freezing and thawing affected the spleen most significantly, and affected all cell types examined by causing a significant increase in DNA damage levels (Table 1). We conclude that since basal levels of detectable DNA damage are affected by cryopreservation of tissue samples, tissues preserved in a similar manner that have real DNA damage induced by any of a variety of means will express exaggerated levels of DNA damage, and that it would be difficult to obtain meaningful comparisons of DNA damage in tissue samples preserved by freezing. 4. References Fairbairn, D.W., O’Neill, K.L. and Standing, M.D. (1993) Application of confocal laser scanning microscopy to analysis of H,OZ induced DNA damage in human cells. Scanning, 15, I36- 139. McKelvey-Martin, V.J., Green. M.L.H.. Schmezer, P.. Poolzobel, B.L.. DeMeo, M.P. and Collins. A. (1993) The single-cell gel-electrophoresis assay (comet assay): a European review. Mutat. Res.. 288, 47-63. Mangan, D.F., Welch. G.R. and Wahl, SM. (1991) Lipopolysaccharide. tumor necrosis factor-alpha, and IL-10 prevent programmed cell death (apoptosis) in human peripheral blood monocytes. J. Immunol.. 146, 1541-1546. Olive, P.L. (1989) Cell proliferation as a requirement for development of the contact effect in Chinese hamster V79 spheroids. Radiat. Res., 117, 79-92. Olive, P.L.. Banath. J.P. and Evans. H.H. (1993) Cell kill-
132
D. W. Fairbairn et al. /Cancer
ing and DNA damage by etoposide in Chinese hamster V79 monolayers and spheroids: influence ofgrowth kinetics, growth environment, and DNA packaging. Br. J. Cancer, 67. 522-530. Olive, P.L. and Durrand. R.E. (1993) Detection of hypoXICcells in a murine tumor with the use of the comet assay. J. Nat). Cancer Inst. 84 (9). 707-71 I. Olive. P.L.. Durrand, R.E. and Banath. J.P. (1990) Detection of etoposide resistance by measuring DNA damage in individual Chinese hamster cells. J. Natl. Cancer Inst.. 82. 779-783. Olive. P.L.. Durrand. R-E.. Banath. J.P. and Evans, H.H. (1991) Etoposide sensitivity and topoisomerase II activity in Chinese hamster V79 monolayers and small spheroids. Int. J. Radiat. Biol.. 60. 453-466. O’Neill. K.L., Fairbairn, D.W. and Standmg. M.D. (1993) Analysis of the single cell gel assay using laser scanning microscopy. Mutat. Res.. in press. Ostling. 0. and Johanson, K.J. (1987) Bleomycin. in contrast to gamma irradiatron, induces extreme variatron of DNA strand breakage from cell to cell. Int. J. Radiat. Biol.. 52(5). 6X3-691. Ostling, 0. and Johanson, K.J. (1984) Microelectrophoretic study of radiation-induced DNA damages in
I2
I3
I4
I5
I6
Lett. 7C (19941 127-132
individual mammalian cells. Biochem. Biophys. Res. Commun.. 123, 291-298. Ostling. 0.. Johanson. K.J., Blomquist. E. and Hagelqvist. E. (1987) DNA damage in clinical radiation therapy studied by microelectrophoresis in stngle tumour cells. Acta Oncol.. 26. 45-48. Ross. K.S.. Haites. N.E. and Kelly, K.F. ( 1990) Repeated freezing and thawing of peripheral blood and DNA in suspension: effects on DNA yield and integrity. J. Med. Genet.. 27. 569-570. Singh. N.P.. McCoy. M.T., Tice. R.R. and Schneider. E.L. (1988) A simple technique for quantitation of low levels of DNA damage in individual cells. Exp. Cell Res.. 175. 184-191. Tice. R.R.. Strauss, G.H.S. and Peters. W.P. (1992) Highdose combinaton alkylating agents with autologous bonemarrow support in patients with breast cancer: preliminary assessment of DNA damage in Individual peripheral blood lymphocytes using the single cell gel electrophoresis assay. Mutat. Res.. 271. 101-113. Vijayalaxmi. Tice, R.R. and Strauss, G.H.S. (1992) A radtation-induced DNA damage in human blood lymphocytes using the single-cell gel electrophoresis technique. Mutat Res., 271. 243-252.
Laser scanning
Letters
76 (1994) 127-132
microscopic analysis in frozen tissues
Daryl William Fairbairn, Richard Van Grigsby, Department
ofMicrobiology. (Received
Brigham
25 October
of DNA damage
Walter August0 Reyes, Kim Leslie O’Neill*
Young University.
1993; accepted
Provo. UT 84602. US.4
8 November
1993)
Abstract
DNA damage is central to research in many fields, especially cancer research and toxicology. The possible loss of DNA structural integrity during freezing or sustained maintenance at low temperatures may present difficulties in the interpretation of data accumulated in studies of tissues collected over a period of time and subsequently evaluated. Using laser scanning microscopic analysis of the recently developed single-cell gel (SCG) assay to measure DNA strand breaks in individual cells, we found that the basal levels of DNA damage in frozen tissue was higher than fresh tissue, but tissues frozen for greater lengths of time do not appear to contain significantly more DNA damage than those frozen for a short period. Evaluation of DNA damage in tumors stored by or collected using cryopreservation may produce artificially exaggerated levels of damage, which could limit analytical interpretations. Key words:
Single-cell
gel assay; DNA breaks:
Laser scanning
1. Introduction DNA
damage
is central
to research
in many
and toxicology. Assays that measure DNA damage are routinely used to determine the effects of chemicals on cells, the capacity of cells to repair induced DNA damage and the potentia response of tumors to chemotherapeutics and radiotherapeutics. Samfields,
especially
* Corresponding
cancer
research
author.
0 1994 Elsevier Scientific 0304~3835/94/$06.00 SSDI 0304-3835(93)03231-S
Publishers
Ireland
microscopy
ples of tissues collected for subsequent DNA analysis are routinely stored at low temperatures. When evaluating the data collected from experiments using such samples, the possibility of DNA degradation or some other alteration in the DNA of stored tissues that could change the profile of DNA damage interpretation is an area of concern. The single-cell gel assay [1 l] is a rapid and sensitive technique for the detection of DNA strand breaks in individual cells. It was originally designed to detect DNA double-strand breaks [lo]. Ltd. All rights reserved
128
Independent modifications of the technique provided a means of detecting DNA single-strand breaks under DNA denaturing conditions [4,14]. Applications for the single-cell gel assay are rapidly expanding into many fields. It was conceived for the determination of DNA damage inflicted with various types of radiation, and was used almost immediately to evaluate tumor responses to radiotherapy in a clinical environment [12]. It was also recognized to have excellent potential in identifying heterogeneous responses within cell populations due to specific subsets of cells. aiding in the identification and treatment of hypoxic cells [6]. which have proved to be significantly more resistant to radiotherapy, as well as the identification of cells resistant to chemotherapeutic agents such as etoposide [5,7,8]. Similar studies may become very useful in predicting tumor responses to various types of therapy on an individual basis (see Ref. 2 for a review of the single-cell gel assay). The effect of freezing and thawing has been examined in peripheral blood lymphocytes using DNA fingerprinting techniques [13] and spectrophotometric analysis of DNA fragments [3]. Both these reports were consistent with a recent report evaluating the effects of cryopreservation on peripheral blood lymphocytes using the SCG assay [l5]. These reports indicate that cryopreservation does not cause a significant increase in the levels of single or double-strand DNA breaks. M’e were concerned that cryopreservation may alter the basal levels of DNA strand breaks in frozen solid tissues. The purpose of this study was to determine the effects of freezing, including repeated freezing and thawing, as well as the effects of the length of storage at low temperatures, on the basal levels of DNA damage in individual cells from frozen tissue using the SCG assay. 2. Materials and methods 2.1. Tissues Male 2.5-week old BALBic mice were used in these experiments. Animals were sacrificed by cervical dislocation, and the spleen, brain, kidney, thymus and liver were immediately removed. Small pieces of tissue, approximately 20 mg each, were frozen to -70°C. Tissues were thawed in a
D. W. Fairhairn et al. /Cancer
Lett. 76 (19941 127-132
37°C water bath and refrozen (at least 1 h elapsed to allow complete freezing of the small pieces of tissue) up to twenty times. Unfrozen control tissues were used immediately upon extraction without freezing. Tissues remained frozen for the same length of time (3-4 days), except where indicated in the long-term freezing experiments (90 days). 2.2. Single-cell gel ussa?’ The SCG assay was performed essentially as described in a previous paper [9]. with some minor modifications. Approximately 20 mg of tissue was homogenized in 2-3 ml of PBS using a Yamoto LSC tissue homogenizer. Cells washed in PBS were suspended in 37°C low melting point agarose at a final concentration of 0.75”/;). A very thin gel was prepared on a fully frosted microscope slide. The slides were placed on ice to allow the agarose to gel and lysed in the dark in 1.0 M NaCl, 0.1% N-lauroyl sarcosine and 0.03 M NaOH for 60 min. The slides were washed in a solution of 0.03 M NaOH and 2 mM EDTA to remove the salt and detergents for an additional 60 min. and electrophoresed in a fresh solution for 25 min at 0.67 V/cm using a Biorad mode1 200/2.0 electrophoresis unit. They were then washed in distilled water and stained with 25 ~1 of 0.2 pgiml ethidium bromide for microscopic analysis. 2.3. Laser scanning microscopy and analysis A Carl Zeiss laser scanning microscope (Thornwood, NY) was used to analyze the comets in all the experiments, as previously described [l]. Cells were identified at 200x magnification and displayed on a Sony monitor by a scanning laser beam from a 5 mW argon laser (488/514 nm). Brightness and contrast were adjusted to clearly define the comet head and tail borders. Measurements were obtained by control of measuring cursors included as part of the microscope system software. Twenty-five comets from each slide were scored. Slides were scored in duplicate, and the experiments were repeated four times. 3. Results and discussion The unique
design
of the single-cell
gel assay
D. W. Fairbairn et al. /Cancer
Lett. 76 (1994)
127-132
30-
129
1+
20
U
frcsb kldaey frozen
kldncy
0 20
A.
40
60
Comet
20
30
C.
Length
fresh
l-+-
40
50
Comet
80
60
100
120
20
(microns)
llvcr
70
Length
B.
40
Comet
60
80
Length
100
120
(microns)
R
80
90
100
(microns)
0 20
D.
100
Co4Aet Le6nogth (ikrdns)
20U
fresh
tbymus
i G TO1
z
20
E. Fig. 1. Response
40
Comet
60
Length
80
100
120
(microns)
of tissue DNA damage
to freezing. (A) brain tissue; (B) kidney tissue; (C) liver tissue; (D) spleen tissue; (E) thymus tissue. Each sample represents 50 comets.
130
D. W. Fairbairn et al. /Cancer
Lett. 76 (1994)
127-132
100
2 i
4o
v
to
0
I brain
0
q
fresh 3 days
2
0
10 Number
kidney
liver
spleen
provides an excellent method for determining DNA strand breaks in individual cells. DNA suspended in agarose forms comets when subjected to an electric field. The comet migration pattern formed during electrophoresis is dependent upon the levels of DNA strand breaks in the cell. The degree of DNA damage is demonstrated in individual comets by the distance of DNA migration in the leading edge of the comet from the body of DNA, which is readily determined by subtracting the resulting comet lengths from unelectrophoresed control lengths. In order to evaluate the effects of freezing as a method of preservation of tissue samples for subsequent DNA damage analysis using the single-cell gel assay, we compared the DNA damage levels in frozen tissue samples with freshly obtained samples. Fig. 1 shows the distribution of comet lengths of both the fresh and frozen tissues for five tissue
freezing
and thawing
of
freeze/thaw
20 repeats
thymus
Fig. 2. Effect of freezingtime on DNA damage. Results of fresh samples are also shown for a comparison of 3-day with 90-day samples, Each bar represents the average of 50 comet lengths.
Table I Effect of repeated
I
60
on DNA migration
Fig. 3. Repeated freezing and thawing increases the levels of detectable basal DNA damage in tissues. Each point indicates the average of 50 comets.
types. There was a significant increase (P < 0.00 1) in basal DNA damage in all tissues examined. Note, however, that the increase in damage was different for each of the cell types. The increased migration ranged from only 11.6 pm for the liver sample to as much as 37.9 pm for the thymocytes. There was also a general increase in the heterogeneity of the comet distribution that occurred with freezing, as the range and standard deviations for the frozen samples were also greater (data not shown). Since we determined that there was an increase in detectable DNA damage in frozen tissues, we were interested in evaluating the effects of increased time spent at low temperatures on DNA basal damage. We compared samples of tissue that had been frozen for 3 days with samples that had been frozen for 90 days (Fig. 2). In all tissues except the liver there was an increase in the migration of
in tissue samples
Sample
Kidney Spleen Thymus
Fresh”
IX”
5x”
IOY”
20x”
7.1 +m 7.0 firn 12.4 pm
39.6 pm 34.7 pm 50.3 pm
44.7 pm 49.5 pm 61.1 pm
46.0 pm 53.8 pm 71.3 pm
50.2 pm 70.8 pm 70.1 pm
The averages of 50 comets are presented. “Distance of migration is determined by subtracting
the unelectrophoresed
control
length
from the comet length
D. W. Fairbairn et al. /Cancer
Lett. 76 (1994)
127-132
DNA with electrophoresis in the samples that had been frozen for the longer period. However, the increase was not statistically significant in the kidney (0.9 pm increase), spleen (0.8 pm increase) or thymus (2.8 pm increase). There was a significant (P c 0.01) increase in the migration (7.6 pm) of the sample frozen for a longer period only in the brain tissue. These results again demonstrate the heterogeneous effects of freezing, or the tissuespecific response to freezing, on the basal levels of DNA damage within the cells of given tissues. It does not appear that increased freezing time is correlaied with increased DNA damage in the individual cells of tissues. The third problem we examined was the effect of repeated freezing and thawing of tissues on DNA damage levels. Fig. 3 illustrates the results of our experiments. Freezing and thawing up to twenty times increased the comet length during electrophoresis by 10.6 pm in the kidney samples, 46.6 pm in the spleen and 20.0 ym in the thymus samples. We compared the lengths of unelectrophoresed control samples with repeated freezing and thawing in order to determine the distance of migration. The distance of migration was determined by subtracting the comet length from the length of the unelectrophoresed controls. There was some migration in the fresh samples, but the migration in the frozen samples was significantly greater. as previously mentioned (Table 1). The distance of migration increases with repetition of the freezing and thawing procedures (Table 1). The increased DNA migration due to this repetition was greatest in the spleen, where the migration distance increased from 24.2 pm in the samples frozen once to 70.8 pm in the samples frozen twenty times. As the single-cell gel assay continues to expand in application into new fields, more attempts will be made to evaluate the DNA damage levels and repair capacities of increasing cell and tissue types that have been exposed to a wide range of chemical, physical and physiological phenomena. Undoubtedly tissues and cells preserved for analysis will be evaluated. In fact such attempts have already been made. A paper evaluating the effects of high-dose combination alkylating agents with autologous bone-marrow support on the peri-
131
pheral lymphocytes of patients with breast cancer compared the effcts of cryopreservation of the lymphocytes on DNA damage levels, and found no increase in DNA migration due to freezing in these cells [ 151. The same group produced another paper later that examined radiation-induced damage. Again, cryopreserved peripheral lymphocytes were used [ 161. Our results indicate that there is a difference in the basal levels of DNA damage between fresh and frozen tissue samples. We found that of the five tissues evaluated. the greatest increase in detectable damage was demonstrated by the thymocytes (Fig. 1). The brain responded to increased length of time at low temperatures with the greatest increase in damage when compared with tissues frozen for a shorter period. None of the other tissues appeared to be affected by increasing the length of freezing time on expression of DNA damage (Fig. 2). Repeated freezing and thawing affected the spleen most significantly, and affected all cell types examined by causing a significant increase in DNA damage levels (Table 1). We conclude that since basal levels of detectable DNA damage are affected by cryopreservation of tissue samples, tissues preserved in a similar manner that have real DNA damage induced by any of a variety of means will express exaggerated levels of DNA damage, and that it would be difficult to obtain meaningful comparisons of DNA damage in tissue samples preserved by freezing. 4. References Fairbairn, D.W., O’Neill, K.L. and Standing, M.D. (1993) Application of confocal laser scanning microscopy to analysis of H,OZ induced DNA damage in human cells. Scanning, 15, I36- 139. McKelvey-Martin, V.J., Green. M.L.H.. Schmezer, P.. Poolzobel, B.L.. DeMeo, M.P. and Collins. A. (1993) The single-cell gel-electrophoresis assay (comet assay): a European review. Mutat. Res.. 288, 47-63. Mangan, D.F., Welch. G.R. and Wahl, SM. (1991) Lipopolysaccharide. tumor necrosis factor-alpha, and IL-10 prevent programmed cell death (apoptosis) in human peripheral blood monocytes. J. Immunol.. 146, 1541-1546. Olive, P.L. (1989) Cell proliferation as a requirement for development of the contact effect in Chinese hamster V79 spheroids. Radiat. Res., 117, 79-92. Olive, P.L.. Banath. J.P. and Evans. H.H. (1993) Cell kill-
132
D. W. Fairbairn et al. /Cancer
ing and DNA damage by etoposide in Chinese hamster V79 monolayers and spheroids: influence ofgrowth kinetics, growth environment, and DNA packaging. Br. J. Cancer, 67. 522-530. Olive, P.L. and Durrand. R.E. (1993) Detection of hypoXICcells in a murine tumor with the use of the comet assay. J. Nat). Cancer Inst. 84 (9). 707-71 I. Olive. P.L.. Durrand, R.E. and Banath. J.P. (1990) Detection of etoposide resistance by measuring DNA damage in individual Chinese hamster cells. J. Natl. Cancer Inst.. 82. 779-783. Olive. P.L.. Durrand. R-E.. Banath. J.P. and Evans, H.H. (1991) Etoposide sensitivity and topoisomerase II activity in Chinese hamster V79 monolayers and small spheroids. Int. J. Radiat. Biol.. 60. 453-466. O’Neill. K.L., Fairbairn, D.W. and Standmg. M.D. (1993) Analysis of the single cell gel assay using laser scanning microscopy. Mutat. Res.. in press. Ostling. 0. and Johanson, K.J. (1987) Bleomycin. in contrast to gamma irradiatron, induces extreme variatron of DNA strand breakage from cell to cell. Int. J. Radiat. Biol.. 52(5). 6X3-691. Ostling, 0. and Johanson, K.J. (1984) Microelectrophoretic study of radiation-induced DNA damages in
I2
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