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INT. J. RADIAT. BIOL ., 1991, VOL . 59, NO. 4, 951-962
Nucleoid sedimentation analysis of DNA superstructure,y-radiation-induced damage and repair in human andchacma baboon (Papia ursinus) peripheral lymphocytes
W. K. A. LOUWt, E . J. VAN RENSBURG$, H. IZATT$and R. I. ENGELBRECHT$
tAtomic Energy Corporation of South Africa Limited, PO Box 582,Pretoria 0001, South Africa$AEC Institute for Life Sciences, University of Pretoria, PO Box 2034,Pretoria 0001, South Africa
(Received 29 July 1990 ; revision received 8 November 1990 ;accepted 14 November 1990)
The use of the chacma baboon (Papio ursinus) in radiobiological investigationsjustifies special attention because it answers many of the criteria of parallelismto the human . The present study was undertaken to establish whether in vitroy-radiation effects in chacma baboon and human lymphocytes are comparable .The sensitive and rapid nucleoid sedimentation technique was employed toevaluate in vitro DNA superstructure, damage and repair in readily obtainableradiosensitive peripheral lymphocytes . Dose-response curves after 60Coy-irradiation were obtained, and by applying single-hit kinetics of the targettheory, an estimation of molecular masses of the supercoiled domains was made .The baboon and human lymphocytes produced analogous results, while anethidium bromide intercalation study also revealed a similarity in average DNAsuperhelical density . Lymphocyte DNA repair after 0 . 5-40 Gy y-irradiationand repair times from 0. 5 to 5 .0 h were evaluated . The repair data obtained frombaboon and human cells after 2 .0 Gy irradiation compared favourably in extentof DNA repair as well as the profiles of the kinetic curves . These findingsindicate that the chacma baboon would be a useful and relevant model forfurther in vivo radiobiological studies on lymphocytes . The effects of sedimen-tation conditions and advantages of using vertical-tube rotors in the nucleoidsedimentation technique are also discussed .
1 . IntroductionAnalysis of DNA damage and repair in readily obtainable radiosensitive
peripheral blood lymphocytes with the sensitive nucleoid sedimentation technique,makes it attractive for the study of genotoxic agents ; for example, ionizing radiationon humans. This method is based on the lysis of isolated lymphocytes in thepresence of a non-ionic detergent and high salt concentrations to release nucleoids(histone-depleted nuclei), followed by rate zonal centrifugation in neutral sucrosegradients . In nucleoids DNA is supercoiled and compact; consequently theysediment more rapidly than their counterparts containing relaxed or unfoldedDNA due to, e.g ., strand breaks (Cook and Brazell 1976b) .
In our previous work we succeeded, by using a vertical rotor, and by theincorporation of the DNA binding dye Hoechst 33258 in the gradients, in changingnucleoid sedimentation into a remarkably effective method for the rapid determin-ation of DNA damage and repair, whereby radiation doses as low as 0 .05 Gy can beestimated (Pitout et al . 1982a,b 1984) .
0020-7616/91 $3 .00 © 1991 Taylor & Francis Ltd
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This sensitivity, together with the lingering restoration of nucleoid sedimen-tation rates after irradiation of human lymphocytes, gave rise to our investigation ofthe possibility of applying this method for retrospective biological radiationdosimetry (Pitout et al . 1982a,b 1983, Van Rensburg et al . 1987, Louw et al. 1988) .Further studies on DNA damage and repair behaviour of B and T lymphocytesubpopulations revealed such similarities, e .g. for dosimetric purposes the tediousseparation of these cells is unnecessary (Pitout et al . 1983, Van Rensburg et al. 1985,1987) .
Since immediate onset of postirradiation DNA strand break repair (in vivo andin vitro) could impair, inter alia, the retrospective dosimetric value of thisprocedure, subsequent investigations revealed that in vitro repair could be elimi-nated by immediate cooling of blood samples to 0°C followed by lymphocyteisolations at 0-4° C, while calibration curves can be utilized to correct for in vivorepair (Louw et al . 1988) .
In view of the fact that in vivo experimental studies of human lymphocyte DNAdamage and repair are beyond dispute, the unsatisfactory simulation of in vivoconditions with whole blood for the construction of these curves is unavoidable .The only alternative is the use of experimental animals, followed by an equally riskyextrapolation to the human, which is confounded by the many anatomical,physiological, and biochemical differences among the various mammalian species(Slaga 1988, Kligerman et al . 1988) . For example, there are differences in DNArepair strategy between rodents and primates . In vitro rodents have a relatively lowcapacity for excision repair, a short lifetime and a high rate of genome evolution,while primates and humans have a high capacity for excision repair, similar DNAstrand break rejoining kinetics, a long lifetime and a low rate of genome evolution(Bohr et al. 1987, Mellon et al . 1987, Breimer 1988, Guedeney et al. 1989) .
An understanding of species differences in, for example, DNA damage andrepair are thus of great value in the validation of animal models for humans .
The use of primates such as baboons (Papio spp .) in these investigations justifiesspecial attention, as they have been used extensively in medical research, andanswer many of the criteria of parallelism to the human (Fridman and Popova1988a,b) .
As chacma baboons (Papio ursinus) are available at reasonable cost in SouthernAfrica, rapid analysis of DNA damage and repair in their readily obtainableperipheral blood lymphocytes, using the sensitive nucleoid sedimentation tech-nique, makes the use of these animals attractive for in vitro and in vivo radio-biological studies .
In the present study we employed this technique to investigate whether thereexist any differences between human and baboon lymphocyte DNA superstructure,as well as cellular responses in respect of DNA damage and repair after 60Coy-irradiation .
2 . Materials and methods2 .1 . Isolation of lymphocytes
Human and chacma baboon lymphocytes were isolated from 30-40 ml lithium-heparinized venous blood . Human blood samples were collected from four healthyadult donors of both sexes . Baboon blood was obtained randomly from a pool of 84healthy adult animals of both sexes, of average weight (± 25 kg) . The baboons were
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housed in outdoor cages and fed a nutritionally balanced diet . Blood samples wereobtained by venepuncture after anaesthesia with ketamine hydrochloride (Ketalar,Parke Davies) (10 mg kg -1 ) and no animal was used more than once a week .
Lymphocytes were isolated under sterile conditions using a Ficoll-Paque(Pharmacia Fine Chem ., Uppsala, Sweden) gradient centrifugation technique(Boyum 1968). The heparanized blood was diluted with an equal volume of Hanks'balanced salt solution (Flow Laboratories) and 10 ml of the mixture was layered onto a Ficoll-Paque gradient (4 . O ml) . The gradients were centrifuged at 800g for 15min at 20°C. Lymphocytes were subsequently harvested from the resultinginterface and washed twice with isotonic saline, (0 .9% NaCl, pH 7 .4) suspended inisotonic saline, counted in a Coulter counter (Model ZB), and the cell suspensionswere adjusted as desired .
2.2 . Nucleoid sedimentationThe method employed was essentially that described by Cook and Brazell
(1975) as adapted by Pitout et al . (1982a). All operations were carried out atconstant temperature (20°C). Stock solutions were treated with diethylpyrocarbo-nate (Sigma Chemical Corp., Missouri) to eliminate possible nuclease and proteaseactivities, and solutions were filtered through Sartorius filters (0 .45 µm pore size,Sartorius, Gmbh, Gottingen) .
Isokinetic sucrose gradients (sucrose for density gradient ultracentrifugation,ribonuclease and deoxyribonuclease free, Merck Chemicals) were prepared inpolyallomer tubes (Beckman Instruments) by means of a Beckman gradient former,and these gradients (linear 15-30% w/v sucrose, pH 8 .0, 37 ml) contained 1 .95 MNaCl, 0 .01 M Tris, 0 .001 M EDTA-Na2 and 1 pg Hoechst 33258 dye (Riedel-deHaen Lab. Chem.) per ml gradient solution . The concentrations of the sucrosesolutions were determined by means of an Abbe refractometer (Kaneko, Tokyo) .For the release of the nucleoid fractions, lysis mixtures (0 . 3 ml) were layered on topof the sucrose gradients . These lysis mixtures contained NaCl, EDTA-Na2 , Trisand Triton X-100 (Boehringer Mannheim, especially purified for membraneresearch) in amounts which, on addition of the lymphocyte suspensions (0 . 1 ml ;0 .5-1 x 106 cells), gave final concentrations of the constituents of 1 . 95, 0 . 1, 0 .002Mand 0 .5%, respectively .
The Triton X-100 was dispensed in small vials and stored under nitrogen at±0°C until just before use. As the Triton X-100 tends to separate from the aqueouslysis mixture on standing, the mixtures were prepared freshly and stirred gentlywith a magnetic stirrer until application on top of the gradients, just prior to theaddition of the lymphocyte suspensions. Twenty minutes after the addition of thecells to the lysis mixtures they were centrifuged in a JV 20 vertical-tube rotor in aBeckman J2-21m centrifuge at 35 800g for 76 min at 20 °C . Gradients were analysedby illumination with 366 nm ultraviolet light, and the position of the blue-greenfluorescent DNA-dye complex was marked. Sedimentation distance was measuredfrom the top of the gradient to the centre of the band . For measurement of DNAsupercoiling, ethidium bromide gradients were prepared as above with the additionof the appropriate concentrations of ethidium bromide (Sigma Chemical Co ., StLouis) instead of the Hoechst dye . The sedimentation behaviour of the nucleoidswas expressed as the ratio of the sedimentation distance of nucleoids from irradiated(or repaired) cells relative to that of the nucleoids from unirradiated cells .
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2 .3 . DNA repairThe lymphocytes were suspended in RPMI 1640 medium containing 20mM
HEPES pH 7 .4, and supplemented with 10% heat-inactivated foetal bovine serum(Flow Laboratories), and aliquots (0 . 1 ml) were y-irradiated at doses of 0 .5-4 .0 Gy,after which they were incubated for various times (0-5-5-0h) in 1 . 0 ml of the abovemedium at 37 °C in an atmosphere of 5% CO 2 . Prior to centrifugation, the cellswere pelleted (200g, 5 min at 37 °C) and resuspended in isotonic saline (pH 7 . 4) to afinal concentration of 1 x 10 6 cells/0 . 1 ml. The repair process was terminated bylysis, followed by nucleoid centrifugation, and the extent of repair was determinedfrom the sedimentation ratios .
2 .4 . y-IrradiationThe 60Co y-irradiation of isolated lymphocytes was performed in a phantom
under 30 mm water at room temperature (DNA repair studies) or under 30 mm ice(dose-response effects) with a Siemens Gammatron 2 Cobalt 60 therapy unit at asample to source distance of 60 cm, and a dose-rate of 0 .21 Gy min _ 1 .
3 . Results and discussionA rapid and reliable lymphocyte isolation procedure which can be performed at
reduced temperatures to eliminate DNA repair (Louw et al . 1988), if the needarises, is a prerequisite for the use of these cells in combination with nucleoidsedimentation for retrospective dosimetry . Ample cells for eight nucleoid gradients(± 1 .0 x 10 6 cells/gradient) could be obtained within 1 .5 h from 15 ml peripheralblood. These preparations had a cell viability of at least 95 % (trypan blue exclusiontest), contained negligible amounts of red cells and less than 5 % non-lymphocyticcells .
The effect of radiation dose on the sedimentation behaviour of nucleoids fromhuman and chacma baboon lymphocytes is presented in Figures 1 and 2 respect-ively. Cells from both species produced similar nonlinear curves, which compare
1.0
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Figure 1 . Dose-response relationship for y-rays on relative sedimentation rates of nuc-leoids from human peripheral lymphocytes . The distance sedimented by nucleoids isexpressed as a ratio relative to the value obtained from unirradiated nucleoidssedimented in the same centrifugation run . Error bars represent standard deviationsof five to seven independent experiments on lymphocytes obtained from four donors .
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Figure 2 . Dose-response relationship for y-rays on relative sedimentation rates of nuc-leoids from chacma baboon peripheral lymphocytes . The distance sedimented bynucleoids is expressed as a ratio relative to the value obtained from unirradiatednucleoids sedimented in the same centrifugation run . Error bars represent standarddeviations of four to nine independent experiments on lymphocytes obtained ran-domly from a pool of 84 animals .
favourably with those obtained with lymphocyte preparations from humans (Lavinand Davidson 1981, Pitout et al . 1982a, Van Rensburg et al . 1985, Erzgraber andLapidus 1988), and the vervet monkey (Potgieter et al. 1988) .
The sensitivity of nucleoid sedimentation as an indicator of DNA strandbreakage derives from an amplification effect whereby in human lymphocytesfor example, one single-strand break can nullify the supercoiling in an entire5 .5 x 109 Da DNA loop (Van Rensburg et al . 1985). However, as the spectrum ofthe superhelical densities allowed in a given loop of DNA is prescribed by its lengthand degree of topological constraint, a reduced nucleoid sedimentation rate doesnot necessarily demonstrate DNA strand breakage (Mattern et al . 1987) . Due to thenon-ideal behaviour and peculiarities of the movement of nucleoids through linearsucrose gradients, care should also be taken in interpreting (Cook and Brazell 1975)as well as in comparing results obtained from different centrifugation conditions .
Most of the nucleoid sedimentation work has been performed in swinging-bucket rotors . Since the sedimentation volume in these rotors is not sector-shaped,a large fraction of the nucleoids in the sticky nucleoid band make contact with thelateral tube wall, which can impair the senstivity and reliability of this procedure(Weniger 1979), as well as causing damage and/or distension of their nuclearmatrices . In contrast, in the vertical tube rotor the nucleoids are initially confined toa narrow vertical plane near the centripetal wall of the tube, and while they aremoving through the first half of the gradient the walls are divergent and so there areno wall effects. The particle path length is short, hence centrifugation times aremuch less than those of swinging-bucket rotors at the same relative centrifugalforce (Rickwood 1982) . Consequently, centrifugation in vertical-tube rotors mayresult in less damage to the nuclear matrix .
As the DNA in nucleoids extends externally from the nuclear periphery(McCready et al . 1979, Adolph 1980, Roti Roti and Wright 1987), the more
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extended damaged (uncoiled) loops may cause an increased frictional resistancerelative to the compact supercoiled loops (Crawford and Waring 1967) . Thecentrifugal forces acting on the various nuclear components with differing buoyantdensities are also strong enough to disrupt the nuclear matrix (Rest et al . 1986) .These effects could put sufficient strain on the fragile lymphocyte nuclear matrix(Cook and Brazell 1976a) to cause additional distension and damage . They can alsogive rise to `fraying out' of the damaged DNA loops in such a way that only themore stable DNA-matrix associations survive, resulting in longer loop lengthestimations, which also depend on the speed of centrifugation where larger targetsizes (longer loops) are obtained at higher centrifugation speeds (Cook and Brazell1975, 1976b, Hartwig 1980) . Longer centrifugation times give rise to a separation ofproteins from the nucleoids (Cress et al. 1989) while distension of the nuclearmatrix reduces nucleoid sedimentation rate without a loss of supercoiling (Cookand Brazell 1978) . The wall effects, as well as the longer travelling distances inswinging-bucket rotors, may also further enhance these effects. The appliedradiation doses can also cause strand breaks in matrix-associated DNA and RNA,which may further weaken the nuclear matrix (Pitout et al . 1982a, Muller et al.1983, Rest et al . 1986) .
All the above-mentioned effects, together with the different assumptions that gowith the various experimental approaches (Paulson 1988), may be the reason for thesignificantly longer DNA loop sizes generally obtained with nucleoid sedimentationthan those obtained with other methods .
As the DNA supercoils of an individual domain (loop) are relaxed by theintroduction of one single-strand break, the levelling out of the dose-responsecurves are produced when all the loops in the nucleoids have received at least onestrand break and the presence of more than one break per loop will not be registered(Figures 1 and 2) .
The single-hit kinetics of the target theory can thus also be applied to the valuesof these curves for the approximate estimation of the sizes of the supercoileddomains (Lea 1962, Dertinger and Jung 1970) .
Assuming that nucleoids sedimenting at rates represented by ratios of 1 .0 and0 .25 contain DNA, all of which, or none of which, is supercoiled (Cook and Brazell1975) and with an efficiency of 50eV/single strand break, it was calculated that0 .01 Gy produced 1 .248 x 1012 breaks/g DNA . Do values were determined byreading off the dose giving log ratio= -1 .0 and dividing by 2 .3 for each componentof the curves (Figures 3 and 4) and using the relationship :
M Do x 1248x10 12x6.022x1023
an assessment of the molecular mass (M) of the target (supercoiled DNA domain)was made (Table 1) .
For comparative purposes the data were analysed in terms of three target groupswhich revealed similar domain sizes in baboon and human lymphocytes (Figures 3and 4, Table 1), which were of the same order of magnitude as those obtained earlierwith the same experimental approach (Cook and Brazell 1975, Filippovich et al .1982, Van Rensburg et al . 1985) .
If the DNA supercoil domain sizes (loop lengths) obtained with similar celltypes under identical nucleoid sedimentation conditions are compared, it can be
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Dose (Gy)
1
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0w0cc00J
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Figure 3 . Estimation of human lymphocyte supercoiled DNA domain sizes by applicationof single-hit kinetics of the target theory to the data presented in Figure 1 .
seen that the differences between human and baboon lymphocytes (Table 1) areonly slightly larger than those between human B and T lymphocytes (VanRensburg et al . 1985), but significantly smaller than those between mouse thymo-cyte fractions (Filippovich et al. 1982) .
The concentration of ethidium bromide which minimizes the sedimentationrate reflects the degree of supercoiling (Crawford and Waring 1967, Cook andBrazell 1975, 1976a, 1978, Mattern et al. 1987) . This concentration for baboonlymphocytes (4-6 pg ml -1 , Figure 5), is roughly the same as those obtained formixed human lymphocytes (Hartwig 1986) as well as for human B and T
0
1 .8r•
• 1 .7Q-0J T .o
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Figure 4 . Estimation of chacma baboon lymphocyte DNA supercoiled domain sizes byapplication of single-hit kinetics of the target theory to the data presented in Figure 2 .
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Table 1 . DNA supercoil domain sizes of human and chacma baboon peripherallymphocytes
1 .0
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_rn 0.6
0..04)Cr 0.4
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Figure 5 . The effect of ethidium bromide concentration on the relative sedimentation ratesof chacma baboon lymphocyte nucleoids . The distance sedimented by unirradiatednucleoids in the presence of ethiduim bromide is expressed as a ratio relative to thevalue obtained from unirradiated nucleoids in the presence of 1 µg/ml Hoechst 33258sedimented in the same centrifugation run . Error bars represent standard deviationsof four independent experiments on lymphocytes obtained randomly from a pool of84 animals .
DomainsDo(Gy)
Molecular mass(Dalton)
Human a 0.62 7.69 x 10 9b 3 . 29 1 . 46 x 10 9c 7 . 76 6 . 22 x 10 8
Baboon A 0 .67 7 . 2 x 10 9B 3 . 07 1 .57 x 10 9C 9 .84 4.9 x 10 8
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lymphocytes (Van Rensburg et al. 1987), suggesting a similarity in the averagedegree of DNA supercoiling in the nucleoids from these two species .
Figure 6 shows the restoration of chacma baboon lymphocyte nucleoid sedi-mentation ratios following 0 . 5, 1 . 0, 2 . 0, 3 . 0 and 4 .0 Gy 60Co y-irradiation damage
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REPAIR TIME (h)Figure 6 . Restoration of human and chacma baboon lymphocyte nucleoid sedimentation
ratios after various 60Co-y doses and repair times from 0 . 5 h to 5 .0 h. Panels a, b, c, dand e, chacma baboon lymphocytes after irradiation with 0 . 5, 1 . 0, 2 . 0, 3 . 0 and 4 .0 Gyrespectively; panel f, human lymphocytes after 2 . 0 Gy irradiation . The distancesedimented by the nucleoids is expressed as a ratio relative to the value obtained fromunirradiated (control) nucleoids sedimented in the same centrifugation run . Errorbars represent standard deviations of four independent experiments obtained fromfour individuals or animals .
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and subsequent repair at 37 °C from 0 .5 to 5-0h. Biphasic repair curves wereobtained with an attending decrease in the extent of repair with increasing doses .The repair data following 2 .0 Gy irradiation from baboons compared favourablywith those from humans in extent of DNA repair, as well as the profiles of thekinetic curves (Figure 6c and Figure 6f) .
The complex interrelationships that exist between diverse processes that rangefrom the rapid repair of simple and easily accessible lesions, to the slow DNAconformational changes and nuclear protein rearrangements (Bohr et al . 1987,Smerdon 1986, Cress et al . 1989) which are all reflected in the nucleoid sedimen-tation behaviour are most likely the cause of the biphasic character of the DNArepair curves. (Stephens and Lipetz 1983, Van Rensburg et al . 1987) .
Despite these complications, the results presented here depict a surprisingsimilarity between human and chacma baboon lymphocyte DNA superstructure,damage and repair, which provides a strategy for further development of theseanimals as valuable radiobiological models of various radiation effects in humans .For example, valuable information regarding in vivo lymphocyte DNA damage andrepair, as well as the effects of modifying agents on these processes, can be obtainedwithout sacrificing animals, as baboons readily tolerate near total body y-irradiationdoses of 1 .0-2 .0 Gy (Myburgh et al . 1984), which falls well within the sensitivityrange of the modified nucleoid sedimentation procedure . The animals are also bigand robust enough to allow frequent blood sampling .
AcknowledgementsThe authors thank Dr G. Beverley and Mr G . Vermaak, of the H. A . Grove
Research Centre, Pretoria, South Africa, for kindly providing chacma baboonperipheral blood samples, and Erika Oosthuizen for typing the manuscript . Thework was supported by the Atomic Energy Corporation of South Africa Limited .
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