Azzam, E. I., Raaphorst, G. P. and Mitchel, R. E. J.. Radiation-Induced Adaptive Response for Protection against. Micronucleus Formation and Neoplastic ...
RADIATION RESEARCH 138, S28-S31 (1994)
Radiation-InducedAdaptive Response for Protection against MicronucleusFormationand Neoplastic Transformation in C3H 10T1/2 Mouse EmbryoCells E. I. Azzam,*,t G. P. Raaphorstt and R. E. J. Mitchel*,l *Radiation Biology Branch, AECL Research, Chalk River Laboratories, Chalk River, Ontario, Canada, KOJ 1JO; and tDepartment of Biology, University of Ottawa, Ottawa, Canada, KIN 1JO
Azzam, E. I., Raaphorst, G. P. and Mitchel, R. E. J. Radiation-InducedAdaptive Response for Protection against MicronucleusFormationand NeoplasticTransformationin C3H 10T1/2Mouse EmbryoCells. Radiat.Res. 138,S28-S31(1994). We have monitored the end points of cellular survival, micronucleusformationand neoplastictransformationfrequency to assess adaptation to ionizing radiation in the C3H 10T1/2 mouse embryocell system. Plateau-phasecells were pre-exposed to an adapting dose of 0.1 to 1.5 Gy low-dose-ratey radiation 3.5 h priorto an acute challengedose of 4 Gy. No adaptingdose improvedclonogenicsurvivaldetectably,whetherthe cells were plated immediatelyafter the acute exposureor held in plateau phase for 3.5 h before plating. However, all chronic adapting doses resultedin both a reductionin micronucleusfrequencyin binucleate cells and about a twofold reduction in neoplastic transformation frequency per viable cell when cells were subsequently exposed to the 4-Gy challenge dose. Our data suggest that a low-dose-ratepre-exposureto ionizing radiation inducesan adaptiveresponsein C3H 10T1/2 cells, and that this responseenhances DNA double-strandbreak repairwhen cells are subsequently exposed to a second radiation dose. This enhancedrepairappearsto be error-freesince these adaptedcells are also less susceptible to radiation-induced neoplastic transformation.
INTRODUCTION For the purposes of radiation protection, radiation effects are assumed to have no dose threshold and to show a linear dose response, with low-dose-rate exposures resulting in reduced effects by about a factor of two. The effects of sequential doses are assumed to be additive (1). Increasing experimental evidence in human and other mammalian cells, however, shows that exposure of lymphocytes (2) or fibroblasts (3) to doses as low as 0.01 Gy induces a process for the repair of chromosomal breaks which renders these cells less susceptible to chromosomal damage caused by a subsequent exposure. This phenomenon, termed adaptive response, has been found to be dependent upon the adapting dose, dose rate, expression time, culture conditions, pH and stage of the cell cycle (reviewed in 1To whom reprintsrequests should be sent.
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ref. 2). An adaptive response to ionizing radiation has been observed for several end points including chromosome aberrations, chromatid aberrations, sister chromatid exchanges, DNA strand breaks, micronucleus formation, mutations and cell survival. An individual variability in the response has been observed (reviewed in ref. 2). These observations in mammalian cells mirror the evidence for the existence of radiationinducible DNA repair systems in prokaryotes and lower eukaryotes (4). What remains unclear at this time, however, is the effect, if any, that the radiationinduced repair capacity observed in mammalian cells has on the carcinogenic risk of a subsequent radiation exposure. The induced repair capacity could be either error-free or error-prone. Induced error-free DNA repair implies a decreased probability of tumor initiation and hence carcinogenic risk, whereas induced errorprone DNA repair implies a corresponding increase in risk. A model system suitable for the study of the carcinogenic effects of a radiation-induced adaptive response is the C3H 10T1/2 "transformation assay" (5), where nontransformed cells in tissue culture can be transformed into demonstrably malignant cells by exposure to radiation. Using this system, we report here the effects of chronically delivered adapting doses on killing, micronucleus formation and neoplastic transformation by an acute challenge dose. MATERIALS AND METHODS The C3H 10T1/2clone 8 cells were obtained from the American Type Tissue Culture Collection (ATCC, Rockville, MD). Cells in passage8 to 10 were seeded at a density of 2 X 105 cells per 80 cm2 flask (Nunc) containing 25 ml of DF culture growth medium (Gibco) supplemented with 15 m M NaHCO3 and 10% fetal calf serum (Sigma, lot 11H-0915). Cells were incubated at 37?C in 2% CO2. Under these conditions,the pH of the medium was maintained at 7.4. The cells were plated 7 to 8 days prior to irradiationfor these studies using confluent cultures (89% in GO/G1phase as determined by flow cytometry). Immediatelyafter irradiation(or in some cases after irradiationfollowed by a defined holding period at 37?C),cells were trypsinizedand replated to yield approximately300 clonogenic cells per flask. Cell survival was determined by colony formation 8 to 10 days after replating.Platingefficienciesranged from 15 to 30%. Flasks for the neoplastic transformationassay were refed on day 10, and then at 7-day intervals,with DF medium supplemented with 5% heat-inactivated (56?C, 30 min) fetal calf serum and 25 gig ml~1
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RADIATION PROTECTION OF C3H 10T1/2 CELLS gentamicin sulfate (Gibco). The pH of the medium varied between 7.3 and 7.4 throughout the experiment. Monolayers were stained after 45 days of incubation.Neoplastic transformationfrequency was estimated using morphologicalcriteria,with only type II and III foci being counted (5). All cellular adapting doses were from 60Co - rays (Atomic Energy of Canada Limited) at a chronic dose rate of 0.0024 Gy/min, at 37?C.Challenge doses were delivered acutely (2 Gy/min) from a Siemens StabilipanII X-ray machine operated at 250 kV and 15 mA with a 1 mm aluminum filter. For the challenge dose, flasks were removed from the 37?Cincubatorfor the time requiredto deliver the dose at room temperature(2 to 3 min). Incubationsbetween or after the radiationtreatmentswere at 37?C. To assay for micronucleusformation,3.5 x 104 cells were plated in chamber slides (Nunc) in the presence of 1 Lg ml-1 of the cytokinesis inhibitorcytochalasinB. After 48 h incubation at 37?C,the rinsed cells were fixed and stained with acridine orange prior to microscopic examination. Micronucleiwere counted in binucleate cells only. Between 1000 and 5000 binucleate cells were counted. Two different methods were used for data analysis. The experimental mean of the neoplastic transformation frequency and the standarderror of the mean were calculateddirectlyfrom the number of replicateflasks for each data point. However, since neoplastically transformedcells are not contact-inhibited, cells may be dislodged during refeeding and form new colonies. To test for errors arising from this potential problem of reseeding, the mean neoplastic transformation frequency and standard error were also calculated from Poisson analysisof the numberof flasks with no transformants[P(0) = e-), where P(0) representsthe probabilitythat a flask will receive zero transformantswhen the average value per flask is X (5). The tables show neoplastic transformationfrequency per viable cell and per cell at risk.
RESULTS Table I shows the results of three independent experiments measuring the effects of an adapting dose of 0.1 to 1.5 Gy low-dose-rate radiation on the survival and neoplastic transformation frequency of plateauphase cells given a 4-Gy acute challenge dose of X rays. The data indicate that none of the adapting doses survival levels detectably, improved clonogenic whether the cells were plated immediately after the acute exposure or held in plateau phase for 3.5 h. However, the results of all three experiments indicate that a prior chronic adapting dose resulted in about a twofold reduction in the neoplastic transformation frequency per viable cell due to the challenge dose of 4 Gy. Increasing adapting doses (0.1, 0.65 or 1.5 Gy) did not show a clear dose response for decreasing neoplastic transformation frequency per viable cell, all resulting in about the same twofold reduction. By themselves, the 0.1- and 0.65-Gy adapting doses resulted in neoplastic transformation frequencies which were fourand twofold higher, respectively, than background, while the 1.5-Gy adapting dose produced a transformation frequency similar to background. Significantly, the neoplastic transformation frequencies of the combined chronic and acute treatments were consistently lower than those due to the acute treatment alone. Calculation of the Poisson mean from the number of flasks containing no transformants indicated that secondary foci formation due to reseeding was not significant. The twofold decrease in the neoplastic trans-
S29
formation frequency per viable cell of adapted cells was also observed after Poisson calculations. When the cells were not pre-exposed, holding them in plateau phase for 3.5 h after the challenge dose also resulted in about a twofold reduction in the neoplastic transformation frequency per viable cell. This holding effect produced a similar or greater additive reduction when the cells were pre-exposed to the adapting doses prior to the challenge dose (Table I). An exception was observed in experiment III, where the 1.5-Gy adapting dose resulted in an increase in the neoplastic transformation frequency per viable cell expressed during the recovery period after the test dose. However, the Poisson calculation shows that this increase might have been due to reseeding. The results of experiment III show that, unlike the effect of holding after the acute challenge dose, holding of the cells for 3.5 h after the 0.1-Gy chronic adapting dose did not alter the neoplastic transformation frequency per viable cell. Neoplastic transformation frequency expressed in terms of cells at risk also showed a reduction resulting from pre-exposure prior to the challenge dose. When calculated this way, the 0.1- and 0.65-Gy pre-exposure doses further reduced the neoplastic transformation beyond that achieved by holding the cells at 37?C for 3.5 h after the challenge dose. Data not shown indicate a 50% reduction in the frequency of type III foci whenever the cells were exposed to an adapting dose prior to the challenge dose. The data in Table II show the effect of pre-exposure to low-dose-rate radiation on micronucleus formation induced by the 4-Gy challenge dose. A reduction in the frequency of micronucleus formation in binucleate cells was observed in adapted cells in all the treatments in the three experiments, as we reported previously for human fibroblasts (3). Furthermore, the frequency of binucleate cells with more than two micronuclei was reduced (up to 35% reduction) in the adapted cells compared to the cells that were exposed to the test dose only (data not shown). DISCUSSION The data described in this report indicate that, in plateau-phase C3H 10T1/2 cells, a pre-exposure to lowdose-rate ionizing radiation can induce resistance to neoplastic transformation and micronucleus formation resulting from a subsequent acute dose of radiation. These data demonstrate that an adaptive response to ionizing radiation exists in these cells. The absence of an observable adaptive response for cellular survival suggests either that cellular survival changes are too small to be detected reliably in this cell line, or that clonogenic survival is dependent on mechanisms different from or in addition to those leading to the adaptive response which reduced neoplastic transformation and micronucleus formation. Other studies have addressed the question of whether one or more mecha-
AZZAM ET AL.
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TABLE I The Effect of Adapting Doses on Radiation-InducedNeoplastic Transformation
Treatment PEa X (4 Gy) Xy (0.67 Gy) - -+ X y -, X
-+
PE X (4 Gy) X -> Y1(0.1 Gy) -+ X
Percentagesurvival (?SD)
No. of flasks
17.3 (0.4) 26.6 (1.8) 41.3 (2.4) 102.4 (4.2) 23.9 (1.2)
12 12 12 12 12
35.1 (3.1)
12
30.1 (0.10) 23.1 (1.04) 30.0 (3.2) 91.9 (9.7) 27.6 (0.98)
12 13 13 12 12
No. of foci
No. of flasks without foci
ExperimentI 1 15 6 2 10 0
11 0 6 10 4 12
ExperimentII 11 1 0 22 4 11 5 7 3 10
Neoplastic transformationfrequency x 10-3 (+SD) Poisson calculation Per cell at risk Per viable cell 0.27 (0.27) 3.2 (0.33) 1.5 (0.45) 0.5 (0.32) 1.7 (0.43) 6.3 (1.1) 2.0 (0.69) 0.5 (0.35) 2.3 (6.3)
0.05 0.15 0.11 0.08 0.07
-X -31.0
-,2
-
y3 (1.5 Gy) 73 X ,3
X -
PE X (4 Gy) X^1 (0.1 Gy) I t ~ X
-'
YI
-
y2 (0.65 Gy) 2- X
t2 X (1.5 Gy) -3 X
Y3
+73
X -42.6
(3.47)
(1.1)
7
aPE, plating efficiency; X, X irradiation at 2 Gy/min; , incubation at 37C for 3.5 h; y, 60Co y irradiation at 0.0024 Gy/min.
nisms lead to expression of the adaptive response by measuring multiple biological end points (3, 7). The results in general tend to support the presence of multiple mechanisms. However, unrepaired DNA doublestrand breaks generally correlate with cell survival. Since the adaptive response results in a reduction of micronucleus frequency, repair of DNA double-strand breaks is enhanced. The DNA content of radiation-induced micronuclei has been shown to include predominantly acentric fragments as a result of unrepaired DNA double-strand breaks (8). Our data indicate that the reduction in micronucleus frequency correlates
with a reduction in the neoplastic transformation frequency, suggesting that an error-freeprocess of damage repair may be enhanced by pre-exposure to low-doserate irradiation. Dose fractionation has been reported to result in a significant reduction in the neoplastic transformationfrequency per viable cell (9), but other reports indicated that incubation time between split Xray doses led to increased neoplastic transformation (10), suggesting the presence of an error-prone repair process. Clear differences exist between the various experimental protocols. Our experiments were done in density-inhibited cells, while previous experiments
RADIATION PROTECTION OF C3H 10T1/2 CELLS
TABLE II Effect of an Adapting Dose on Radiation-Induced MicronucleusFormation Percentageof binucleatedcells with micronuclei Treatment
Observed(+SD)
73.2 (2.9)
-+
y (0.65 Gy)
3' X y -- X
91.6 80.0
Experiment II 11.5 (0.75) 85.3 (2.3)
Control X (4 Gy) X -
72.4 (2.2)
16.2 (0.73)
7Y(0.1 Gy) -Y
81.5 (2.0) 70.2 (1.5)
X ->
90.0 77.2
7, (0.65 Gy)
19.1 (0.80)
'2 -X 7,2 X
81.9 (2.1)
92.9
72.8 (2.1)
80.0
Y3(1.5 Gy) Y3 X 3 X -
24.0 (1.0) 84.9 (2.3) 70.9 (1.3)
97.8 85.0
Experiment III 5.49 (0.54) 85.1 (4.0)
Control X (4 Gy)
X --
62.4 (2.9)
14.1 (1.2)
y (1.5 Gy) y- X y -, X -
ACKNOWLEDGMENTS
17.0 (1.1)
81.4 (3.2) 63.8 (2.3)
-*
' 1X
ror-free repair. If a similar enhancement of error-free repair were to be generally true in human cells, this result would have important implications for the estimation of cancer risk resulting from multiple exposures to ionizing radiations.
This researchwas supportedfinanciallyby the CANDU Owners Group in Canada and the Natural Sciences and Engineering Research Council of Canada.The authors thank J. Jackson, K. Gale and P. Lee for their assistancein performingthe experiments.
ExperimentI 10.2 (1.3) 84.7 (3.4)
Control Xa (4 Gy) X
Expected
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-
69.3 (3.3) 60.0 (2.8)
93.7 70.9
aX, X irradiationat 2 Gy/min; +, incubation at 37?Cfor 3.5 h; y, 60Co y irradiationat 0.0024 Gy/min.
were done in asynchronous populations. Our experiments also used low-dose-rate adapting doses. Considering the conflicting results, two repair processes may be involved in the repair of potentially neoplastic transforming damage: one error-free and one error-prone. However, our data suggest that an adapting dose delivered at a low dose rate enhanceser-
REFERENCES 1. UNSCEAR, Report to the General Assembly, with annexes. United Nations, New York, 1988. 2. S. Wolff, Low dose exposure and the induction of adaptation. In Low Dose Irradiation and Biological Defense Mechanisms (T. Sugahara, L. A. Sagan and T. Aoyama, Eds.), pp. 21-28. Elsevier Science Publishers,Amsterdam,1992. 3. E. I. Azzam, S. M. de Toledo, G. P. Raaphorst and R. E. J. Mitchel, Radiation-inducedradioresistance in a normal human skin fibroblastcell line. In Low Dose Irradiationand Biological Defense Mechanisms (T. Sugahara,L. A. Sagan and T. Aoyama, Eds.), pp. 291-294. Elsevier Science Publishers, Amsterdam, 1992. 4. R. E. J. Mitchel and D. P. Morrison, Inducible DNA-repair systems in yeast: Competition for lesions. Mutat. Res. 183, 149-159 (1987). 5. C. A. Reznikoff, J. S. Bertram, D. W. Brankow and C. Heidelberger, Quantitative and qualitative studies of chemical transformationof cloned C3H mouse embryo cells sensitive to postconfluenceinhibition of cell division. Cancer Res. 33, 32393249 (1973). 6. A. Han and M. M. Elkind, Transformation of mouse C3H10T1/2cells by single and fractionateddoses of x-rays and fission spectrumneutrons. Cancer Res. 39, 123-130 (1979). 7. J. D. Shadley and G. Dai, Cytogenetic and survival adaptive responses in G1 phase human lymphocytes. Mutat. Res. 265, 273281 (1992). 8. B. M. Miller, T. Werner, H-U. Weier and M. Niisse, Analysis of radiation-inducedmicronuclei by fluorescence in situ hybridization (FISH) simultaneously using telomeric and centromeric DNA probes. Radiat. Res. 131, 177-185 (1992). 9. C. K. Hill, F. Buonaguro and M. M. Elkind, Multifractionation of 60Co gamma-rays reduces neoplatic transformation in vitro. Carcinogenesis5, 193-197 (1984). 10. R. C. Miller, E. J. Hall and H. H. Rossi, Oncogenic transformation of mammaliancells in vitro with split doses of x-rays. Proc. Natl. Acad. Sci. USA 76. 5755-5758(1979).
