INT. J. RADIAT. BIOL., 1984, VOL. 46, NO. 5, 529-540

Detection in situ of y-ray-induced DNA strand breaks in singlecells: enzymatic labelling of free 3'-OH ends

B. FERTIL, S. MODAKt, N. CHAVAUDRA, H. DEBRY,F. MEYER: and E. P. MALAISE

Laboratoire de Radiobiologie des Cellules normales et Cancereuses(Unite Inserm 247), Institut Gustave-Roussy, 94805 Villejuif, France,and tEcole Nationale Superieure des Mines de Paris, Centre de Geostatistique etde Morphologie Mathgmatique, 35, rue Saint-Honor6, 77305 Fontainebleau,France

(Received I February 1984; revision received 17 May 1984; accepted 30 May 1984)

We report a procedure allowing the detection and counting of free 3'-OH DNAstrand extremities in single cells in situ. Terminal transferase (TdT) catalysed theincorporation of 3H-dGMP into fixed nuclei of human colonic adenocarcinomacells (HT29), using free 3'-OH ends as initiator. Radioactivity was detected byautoradiography and determined quantitatively with a rapid image-processingsystem for grain counting. The initiator activity for TdT increases with the doseof y-rays in the dose range 25-20 Gy.

Indexing terms: DNA breakage, human cell line, autoradiography.

1. IntroductionIonizing radiations introduce a variety of lesions into DNA (for an overview, see

Hart et al. 1979), resulting in inactivation of proliferation capacity, mutation and/orchromosomal aberrations. Induction of single or double strand breaks decreases themolecular weight of DNA, which can be measured by sedimentation throughalkaline or neutral sucrose gradients (Brash and Hart 1978), viscoelasticity measure-ments (Kavenoff and Zimm 1973), one-dimensional electrophoresis in neutral oralkaline agarose gels (McDonell et al. 1977) and two-dimensional gel electrophoresis(Modak and Beard 1980). Low dose effects have been mainly investigated withtechniques allowing the measurement of the rate of DNA unwinding in alkali(Rydberg 1975) or employing alkaline elution from membrane filters (Kohn et al.1976).

Most of these techniques deal with radioactively labelled DNA and this createsthe possibility of inducing damage due to radiolysis. In addition, during centrifug-ation, large DNA molecules undergo anomalous sedimentation. DNA can also suffermechanical shear, resulting in the underestimation of the molecular weight of verylong molecules (Kavenoff and Zimm 1973). The sedimentation techniques are thusnot sufficiently sensitive to study the effect of low dose radiation on eukaryotic DNA.Finally, all these methods lead to the estimation of the mean molecular weight ofDNA, from a mixture of cells which may be radiobiologically heterogeneous. For

tPermanent address: University of Poona, Zoology Department, Pune, 411007, India.

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example, proliferating cells possess differential radiosensitivity depending on theirposition in the cell cycle (Sinclair and Morton 1966). So far as survival is related tothe extent of residual strand breaks in DNA (Dugle et al. 1976, Chadwick andLeenhouts 1981), each phase of the cell cycle is expected to be characterized by aspecific dose-effect relationship. The analysis of asynchronous populations by theabove methods will, however, give only mean values. The in situ detection of 3'-OHDNA ends reported in this paper can circumvent these difficulties as radiation effectscan be observed in single cells.

In a series of reports, Modak and co-workers (Modak et al. 1969, Modak andBollum 1970, Modak 1972, Modak and Traurig 1972) showed that terminaldeoxynucleotidyl transferase (TdT) from calf thymus catalyses the synthesis ofradioactive polydeoxyribonucleotides in fixed cell nuclei, using endogeneous free 3'-OH ends as initiators. Using this technique, the number of 3'-OH ends has beenshown to increase during the natural degeneration of cell nuclei (Modak and Bollum1970, 1972, Modak 1972, Modak and Traurig 1972). Ionizing radiations createstrand breaks in DNA and at least a portion of this breakage generates free 3'-OHends (for review, see von Sonntag et al. 1981). Using this in situ procedure, we reportthat TdT-catalysed incorporation increases with doses of y-rays up to 20 Gy in ahuman cell line.

2. Materials and methodsThe human colonic adenocarcinoma cell line HT29 has been used extensively for

radiosensitivity studies with the culture conditions described by Fertil et al. (1980).Major steps of experimental protocol are shown in figure 1. Cells were grown asmonolayers on glass slides in culture chambers (Flaskette, Lab-Tek). Exponentiallygrowing sparse cultures were irradiated with 2'5, 50, 7-5, 10 or 20 Gy of y-rays from a13 7Cs source (dose rate 0-85 Gy/min, room temperature). Cells were washed in 0-9per cent NaCl and fixed in methanol: acetone (1: 1, for 30min at -20C)immediately after irradiation so that endogeneous DNA polymerases were inacti-vated and post-irradiation DNA repair was maximally prevented. Slides were thenair-dried and treated with 001 N HCI for 15 min on ice to denature DNA in situ so asto make all 3'-OH ends available as initiators (Modak et al. 1969). The slides wereextensively washed in distilled water, dehydrated in a graded series up to 80 per centethanol and air-dried. Enzyme incubation wells were mounted on the slides aspreviously described (Modak et al. 1969, Modak and Bollum 1970) using paraffin(m.p.=45°C) as adhesive.

The enzyme reaction mixture contained 85 pCi 3 H-dGTP/ml (specific activity13 Ci/mM, Radiochemical Centre, Amersham), 415 units/ml of terminal transferasefrom calf thymus (Boehringer-Mannheim), 0-85 mM dithiothreitol, 17 mM HEPES(pH 7'4), 085 mM MnCl 2 , 725 per cent glycerol and 0008 per cent bovine serumalbumin.

The overall procedure for the treatment of fixed cells with the enzyme reactionmixture is similar to that described elsewhere (Modak and Bollum 1970, 1972).Fifty-five microlitres of reaction mixture was added to each well, which was thensealed with a coverslip and incubated for 30min at 37°C. After removal of thecoverslip, the reaction was stopped by rinsing the slide with ice-cold 0-1 5 M NaCl;the incubation well was discarded afterwards. The slide was then washed once with 5per cent trichloroacetic acid (TCA) containing 1 per cent inorganic pyrophosphate

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l}3'OH $'P

iP 3 ' OP H

DNA denai.ti .. |ti. $|

3'OH

P ;o

| ~~~~~3'OH

| uadioaphy| flg

| utatic rain 3cann in _

Figure 1. Protocol.

(PPi) for 30 min, on ice and twice with 5 per cent TCA alone. TCA was removed bywashing the slides in running water for 15 h. They were then dehydrated through analcohol series and washed in xylol in order to remove the adhering paraffin. Slideswere then rehydrated down to 80 per cent ethanol and air-dried. For autoradio-graphy, slides were dipped in Ilford L-4 emulsion diluted 1: 1 with distilled watercontaining 1 per cent glycerol. Slides were dried and exposed in darkness for 9-24 hat 4°C. Autoradiographs were developed in Kodak D-19B and fixed in AL4. Cellswere lightly stained with Meyer's haemalun, dehydrated and mounted in Eukitt.

Slides were examined under oil-immersion optics. Grain counts were performedon a TAS texture analyser from Leitz. Details of the processing method andalgorithm are explained in the accompanying Appendix. Basically, the microscopeimage was transmitted to a video screen (256 x 256 pixels). The outline of eachnucleus on the video screen was traced with a light pencil and the number of grainswas determined automatically using the 'Top Hat' transformation procedure and bytaking into account the probability of grain-overlap. The number of backgroundgrains was estimated in regions adjacent to cells. The number of grains per nucleusor per unit nuclear area of both control and 7-irradiated cells was estimated and thebackground count equivalent to each nuclear area was automatically subtracted.

Grain counts are presented in the form of histograms as well as cumulativefrequencies. Geometric means are used throughout this paper.

3. ResultsIn the present experimental conditions, the nuclear area is specifically labelled

(figure 2). Cells incubated in the presence of radioactive reaction mixture withoutenzyme exhibit a radioactivity equivalent to that of background. This proves that thelabelling is specific for the sites recognized by the enzyme.

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The autoradiographs of HT29 cells show that this labelling increases with thedose of y-rays (figure 2). This is illustrated and quantitated in figure 3, wherehistograms of grain count/nucleus and grain count/unit nuclear area are presentedfor both 200 control cells and 200 irradiated cells (75 Gy). Thus, the mean numberof grains/nucleus increases from 128 (119-139) in control to 258 (241-277) inirradiated cells (95 per cent confidence intervals). Similarly the grain count/unitnuclear area increases from 70 (6-6-75) in control to 15 (14-16) in irradiated cells. Inaddition to increased mean grain number, the labelling distribution widens. Takinginto account variation of labelling among cells for a given slide, it was decided toexamine 50 nuclei/slide for subsequent grain counting. The computation ofskewness and kurtosis (gl, g2 ) shows that the hypothesis of log-normal distributionis valid (with 95 per cent confidence) for over 75 per cent of all histograms analysed.This enabled us to plot the data as probit of cumulative frequency of nuclei against

Figure 2. Labelling intensity of unirradiated HT29 human adenocarcinoma cells and thoseirradiated with 25, 5, 75 and lO 0Gy.

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F j---I --

t'-�j-1

u-,- t ' ' I ' · _ ,'If' I I7.5-Gy

30 -

20-

10-

0 200 400 600 0 10 20 30

GRAINS / NUCLEUS GRAINS/UNIT NUCLEAR AREA

Figure 3. Typical distribution of the number of grains per nucleus or per unit nuclear areafor an unirradiated and an irradiated (75 Gy) HT29 cell population.

the logarithm of grain count/nucleus, whereby log-normal distributed data shouldappear as a straight line. The experimental data corresponding to one experiment aregiven as an example (figure 4). These data allow the above statement to be verified,with the exception of about 30 per cent of control nuclei with low grain counts. As afirst approximation, the geometrical mean can be used to reflect the effect ofirradiation, more especially as the increase in dose essentially results, in thesecoordinates, in a translation of cumulative frequency lines towards higher labellingintensities.

We examined the grain count data gathered from five different experiments(table), among which the absolute grain counts varies as much as fourfold as a

.95

.9

I

a' .7

.5

.1

I .I

.DS

I0

*0 *l

I* 0

* i

0 ou .

0 *

0so 100

GRA INS/NUCLEUS

Soo 1000

Figure 4. Cumulative frequency distribution of the number of grains per nucleus forincreasing dose of y-rays (one typical experiment): * control, 0 25 Gy, * 5 Gy, [l75Gy, 1OGy.

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Absolute mean grain count with 95 per cent confidence limits and mean grain count relative to thecorresponding control are presented as a function of dose for five experiments.

Mean grain count

Experiment 0 Gy 2-5 Gy 5Gy 75 Gy 10 Gy 20 Gy

1 106(86-130) 136(119-157)176(154-201)219(192-252)226(201-254) -1 128 1-66 207 213

2 25(21-30) - 52(44-62) - 58(51-66) -1 208 232

3 31(24-40) 32(28-37) - 67(57-79) 74(65-83) -1 1-03 216 239

4 56(50-64) - 101(84-121) - - -1 180

5 26(20-31) - -- 123(104-146)137(107-176)1 473 527

function of exposure time of the emulsion. Nevertheless, the ratio of mean graincount for nuclei irradiated at any dose to that of corresponding control nuclei do notdepend on mean labelling intensity (table). The relative mean grain count/nucleusplotted against the dose of y-rays falls on a straight line (figure 5), with a correlationcoefficient of 086 (P< 001). This linear regression extrapolates to 06 (this value isnot statistically different from 1) for dose 0.

-

w

g=

J

:

W~

I4

DOSE (Gy )

Figure 5. Relationship between the dose of ?-rays and mean grain counts/nucleus.

4. DiscussionCalf thymus terminal trasferase requires an initiator in the form of DNA with a

free 3'-OH end preceded by a single strand at least three nucleotides long to catalyseend-addition synthesis of polydeoxyribonucleotide homopolymer (Bollum 1960,1962, Bollum et al. 1964, Yoneda and Bollum 1965, Kato et al. 1967). This reaction islinear with time in the presence of excess enzyme and excess substrate. The enzymerecognizes DNA initiators in fixed cell nuclei (Modak and Bollum 1970, 1972) inwhich the incorporation of radioactive deoxyribonucleotide monophosphates can be

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detected autoradiographically. Silver grains can be counted optically and theirnumber was shown to increase linearly with increased enzyme reaction times(Modak and Bollum 1972). In the present experiments, we found that subjectingcultured cells to increasing doses of y-rays produced an increasing quantity ofinitiator molecules (free 3'-OH ends) which react with TdT.

y-Rays are known to induce breaks in DNA, some of which involve thephosphodiester bond, manifested by the appearance of 5'-P and 3'-OH extremities.In our experiments, DNA was denatured in situ by acid treatment so that all 3'-OHends were rendered accessible as initiators for TdT (Modak et al. 1969, Modak andBollumrn 1970, 1972). All 3'-OH single strand breaks are thus potentially detectable.In these conditions double strand breaks provide two initiator sites. It could beargued that fixation and/or acid treatment could interact with other radio-inducedeffects, in such a way that an increase of 3'-OH labelling with the dose could result.For instance, DNA relaxation induced by low doses (Cook and Brazell 1975) couldgive increased numbers of fixation- and/or acid-induced breaks or affect theaccessibility of the breaks for the enzyme. Relaxation intensity, however, appears tosaturate after 10 Gy (Cook and Brazell 1975). It does not seem to be the case in ourhands. A saturation effect could nevertheless take place, but at much higher doses(100 Gy) (Modak and Price 1971), in a dose range where relaxation effect is unlikely.Denaturation with HCl could also be expected to result in additional breaks in DNA,which could interact with radiation effects. In fact, a five-fold increase in labellingintensity of unirradiated cells is observed when labelling of denatured DNA iscompared to that of native DNA. This could well be explained on the basis of thealready present single strand breaks consequently exposed to the enzyme. Fur-thermore, when labelling takes place on native material, the dose effect relationshipwhich is suggested (1-3 increase in labelling intensity for 5 Gy, unpublished results),does not reveal the existence of an important interaction between acid-inducedbreaks and radiation effect. However, as interaction between fixation and irradiation,though unlikely, cannot be ruled out, we cannot assert that a direct measurement ofradio-induced breaks is achieved by our technique, especially as repair, that is knownto take place during irradiation, can reveal and/or remove 3'-OH breaks. Themeasurement of repair efficiency (effect of irradiation at 37°C compared to that ofirradiation at 0°C) which is now under investigation will provide a more definiteanswer. Nevertheless, the dose-effect relationship we observed confirms thatirradiation leads to the appearance of specific sites that react with TdT when they arerendered free.

In terminally differentiating lens fibre cells, nuclei degenerate and disappear witha concomitant loss of DNA (Modak and Perdue 1970), an increased initiator activityfor TdT (Modak and Bollum 1970, 1972, Modak 1972) and an increased templateactivity for DNA polymerase (Modak et al. 1969). These results were interpreted asshowing that in situ techniques could demonstrate DNA strand breaks in single cells.Independent biophysical determinations of DNA molecular weights in a populationof lens cells (Piatigorsky et al. 1973, Appleby and Modak 1977, Counis et al. 1977)supported this interpretation, just as the present results also strongly support it.

It must be pointed out that even control cells are labelled. This in not surprisingas, in addition to the 3'-OH extremities of DNA molecules, it has been shown that upto several hundred (700-2000) single strand breaks may be present in physiologicallyactive cells (Ahnstr6m and Edvardsson 1974). At least a proportion of these breaksand extremities is available to the enzyme. Under our conditions, one grain roughly

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reveals the incorporation of 3 x 105 nucleotides. Thus, assuming that 1000 sites arelabelled in a control cell where 50 grains are scored, the mean string length isexpected to be 15 x 104 nucleotides. Such a value is not unlikely when initiator sitesare present (Kato et al. 1967). Grain number distribution from cell to cell alsodeserves some comments: breaks are probably not evenly distributed among the cellssince S phase cells are believed to contain between 5000 and 10000 breaks pergenome (Ahnstr6m and Edvardsson 1974) and DNA strand number varies duringthe cell cycle. This cellular heterogeneity can likely account for part of the broadnessof the grain number distribution, although this remains to be demonstrated. We haveobserved that 75 per cent of the grain number destributions can be considered as log-normal. This is in fact generally expected when numerous biological and inherentexperimental factors are involved and/or the observable quantity arises by asuccession of steps (Koch 1966, 1969).

In the present study, a dose-effect relationship was demonstrated for doses up to20 Gy (figure 5). The regression line passes through the origin so that the dose-effectrelationship is apparently linear. A single strand break is considered to be the resultof a single event related to the passage of a particle and thus proportional to the dosedelivered (Kellerer and Rossi 1972, Chadwick and Leenhouts 1973). This has beenobservedexperimentally with other models and other methods (e.g. Ono and Okada1974, Lennartz et al. 1973), in conditions such that DNA repair did not occur. Thepresent experiments were performed at 20°C. In these conditions, the half-life ofDNA lesions varies from 10 to 40min (Chadwick and Leenhouts 1981). Since ourcells were fixed only at the end of the irradiation, repair was expected to take placeduring the irradiation and occur to a greater extent with increasing time ofirradiation (10Gy required 12min to deliver). Nevertheless, the precision of ourexperimental results and the dose range used do not enable us to conclude firmly onthis point.

The measurement of the doubling dose is one of the original features of ourmethod. It can be observed that a dose of 7'5 Gy creates about as many 3'-OH breaksas are found in control cells. Based on Kellerer's calculation (Kellerer 1979), we haveestimated that 7'5 Gy would create 10 000 single strand breaks in an HT29 cell. Onan other hand, control cells contain 700-2000 strand breaks (Ahnstr6m andEdvardsson 1974). Our observations thus indicate that 3'-OH breaks are anappreciable component of y-rays-induced strand breaks, so far as an importantproportion of spontaneous breakage in a physiologically active cell release 3'-OHends.

The TdT method thus results in the demonstration of single strand breaksbetween 3'-OH and 5'-P of DNA in situ in single cells in a manner which allowsdetection of damage inflicted by doses as low as 25 Gy. With the use of radioactivedeoxyribonucleotides of higher specific activity, longer Putoradiographic exposuretimes and an automatic grain counting procedure with a high resolution image, thesensitivity of this method should be increased substantially. It has been shown thatthe kinetics of TdT-catalysed incorporation is linear with time of incubation andthe slope of the labelling curves apparently increases with the number of free 3'-OHends in situ (Modak and Bollum 1972). The measurement of the kinetics ofTdT-catalysed incorporation in irradiated cells will increase the sensitivity of thismethod still further.

One of the most important results of the availability of this method is that it ispossible to analyse the radiation effects on a cell to cell basis. The automatic image-

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processing system that we used to count grains in this study also leads to thesimultaneous measurement of nuclear areas and can be readily exploited for theconcomittant estimation of DNA amount per nucleus, nuclear area and the graincounts. This will enable us to study the relationship between accumulated DNAdamage (unrepaired or non-reparable) and the radiosensitivity of cell subpopu-lations. Another interest of this method could be the measurement of high l.e.t.radiation induced breaks. In our hands, a cluster of breaks will be analysed as severalbreaks while other techniques, such as the unwinding method or alcaline elution, willdetect it as one break.

5. Appendix: Automated grain countingAutomated grain counting continues to be an important field of technical

research (Sklarew 1982 b). The major problem in the image analysis of autoradio-graphs of cells by black and white video systems is the discrimination among variousshades of grey: it is very difficult to distinguish between real silver grains (black) anddarkly stained regions of nuclear and nucleolar chromatin in the darker regions.Furthermore, in densely labelled autoradiographs, the grain clustering effectconsiderably decreases the resolving power (for reviews see Sklarew 1982 a, b). Themethod we have used is based on various tools developed at the Centre deMorphologie Mathematique in Fontainebleau and has been designed by F. Meyer toovercome these difficulties. (An extensive course in mathematical morphology canbe found in Serra (1982)). The same group developed also the Texture AnalysisSystem (TAS) (see Klein 1976) now manufactured under licence by Leitz.

There are two phases in the determination of the number of silver grains: aproper segmentation of the image in order to visualize the grains; and the counting ofthe grains.

(a) Segmentation of the grainsIn our experiments, the background of the nuclei is relatively heterogeneous,

with dark nucleoli in the centre (Figure 6 A, left panel). If one regards the grey toneimage as a topographic surface, the silver grains would appear as small peaks on a lowfrequency variation representing the background, as illustrated in figure 6 B, rightpanel and 6 C, right panel.

It is often quite impossible to find a threshold in the image, which would detect allthe grains and only the grains. This is illustrated in figure 6 B (furthermore it wouldbe too time consuming to adjust a threshold for each nucleus). On the peaks,however, it is possible to place a small 'hat'. The top of the 'hat' is 'burst', and whatemerges is recognized as silver grains. This transformation is called 'top-hat'transformation (Meyer 1978) and the result is shown in figure 6C. With thistechnique, it is possible to work on an entire slide without readjusting the size of the'hat'.

(b) Counting the number of silver grainsSince the silver grains very often overlap when the nucleus is crowded, a simple

counting of the number of connected particles would systematically lead to anunderstimation. A good estimation can be obtained by considering the graindistribution as a realization of a Boolean model (Matheron 1967) (grains are

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B

average threshold

C

"top hat" transformation

Figure 6. A (left), Original image as seen by the video camera. A (right), Investigated area. B(left), 'Best' threshold: not all grains are detected, but the nucleoli are already seen. B(right), Grey tone distribution: nucleus, nucleolus and grains. No threshold can detectthe grains. C (left), Result of the 'top-hat' transformation. C (right), Grey tonedistribution: the silver grains are sharp peaks, on which it is possible to place a 'hat'. Thepart emerging from the top of the 'hat' is detected as a peak.

implemented at Poisson points in a field Z, and these grains may overlap). In thismodel, a non-biased estimation can be obtained by:

n=-- xln( -)s E

where n is the number of grains in a nucleus; E is the area of the nucleus; S is the areaoccupied by the grains; s is the mean area of a grain. According to this formula, thisestimation of n requires only area measurements of S, E and mean area of a grain s.This estimation is very robust, and gives good results even when the underlyingmodel is not Boolean (Digabel 1977), and/or when the number of grains is low.

We have verified that the estimation of the number of grains from the surface theyoccupy gave a figure identical to that obtained by direct estimation, Also, the mean

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size of a grain was obtained from these observations. The validity of the Poissonianapproximation of grain distribution was verified by the fact that the threeindependent experiments presented in this report led to the same dose-effectrelationship, even though mean labelling among experiments was very different(mean control values varied from 30 to 120).

AcknowledgmentsThe authors would like to acknowledge Miss Mireille Lahon for her skillful

secretarial assistance. This work was supported by the Ligue Nationale FrangaiseContre le Cancer (Comit6 des Hauts-de-Seine) and contract B00-E52/3H 23 77 fromthe Comit6 de Radioprotection de l'Electricit6 de France.

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Theory of Radiation, Monographs on Theoretical and Applied Genetics, 5, edited by R.Frankel, G. A. E. Gall, M. Grossmann, H. F. Linskens and D. de Zeeuw (Berlin,Heidelberg, New York: Springer Verlag), p.21.

COOK, P. R., and BRAZELL, I. A., 1975, J. Cell Sci., 19, 261.COUNIS, M. F., CHAUDUN, E., COURTOIS, Y., 1977, Der. Biol., 57, 47.DIGABEL, H., 1977, Geometrical Probability and Biological Structures, edited by R. E. Miles

and J. Serra (Berlin, Heidelberg, New York: Springer Verlag), p.331.DUGLE, L., GILLEPSIE, C. J., and CHAPMAN, J. D., 1976, Proc. natn. Acad. Sci. U.S.A., 73,

809.FERTILI, B., DESCHAVANNE, P. J., MALAISE, E. P., and LACHET, B., 1980, Radiat. Res., 82, 297.HART, R. W., D'AMBROslo, S. M., NG, K. J., and MODAK, S. P., 1979, Mech. Ageing Dev., 9,

203.KATO, K. I., GONCALVES, J. M., HAUTS, G. E., and BOLLUM, F. J., 1967, J. biol. Chem., 242,

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