INT .
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RADIAT . BIOL .,
1992,
VOL .
62,
NO .
3, 279-287
Heavy ion-induced DNA double-strand breaks in yeast T. C. AKPAf , K . J. WEBERt§, E . SCHNEIDERt, J . KIEFERt*, M. FRANKENBERG-SCHWAGER¶, R . HARBICH¶ and D . FRANKENBERG¶
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(Received 9 August 1991 ; second revision received 30 March 1992 ; accepted 12 April 1992)
Abstract. DNA double-strand break (dsb) induction in diploid yeast was measured by neutral sucrose sedimentation after exposure to very heavy ions with values of linear energy transfer . Linear (LET) ranging from about 300 to 11500 keV/pm fluence dependencies were found in all cases from which dsb production cross-sections (ad .b ) could be calculated . Corresponding cross-sections for cell killing ((a ;) were derived from final slopes of survival curves measured in parallel and for the same fluence range. A close correlation was found between a ; and a d,b . It is calculated that over the entire LET range, including 30 Me V electron irradiation, about 22 dsb are induced per lethal event when high exposures are considered .
1. Introduction DNA double-strand breaks (dsb) appear to be the most important DNA lesions induced by ionizing radiations related to chromosomal aberrations and cell death (see Frankenberg-Schwager 1990 for a recent summary) . This is normally implied from the close correlations between observed cellular radiation response and measured dsb frequencies - as reported for many different cell lines - and a parallelism in the change of these experimental endpoints when modifying conditions were employed . Accordingly, the influence of radiation quality (i.e. linear energy transfer, LET) on lesion induction and damage expression has found wide attention, but despite numerous studies a conclusive picture is proving to be hard to reach (Todd and Tobias 1974, Kiefer 1985, Kraft 1987, Roots et al . 1990) . The detection of DNA dsb in mammalian cells for a `biologically relevant' dose range (below approximately 20 Gy) is largely hampered by the high residual molecular weights of DNA fragments generated with such doses . Since they may not easily be *To whom correspondence should be addressed . tStrahlenzentrum der Justus-Liebig-Universitat, GieBen, Germany . ¶Institut fur Biophysikalische Strahenforschung der GSF, Frankfurt-Main, Germany. $Present address : Physics Department, Ahmadu Bello University, Zaria, Nigeria . §Present address : Strahlenklinik der Universitat, Heidelberg, Germany .
analysed by the conventional neutral sucrose sedimentation technique at low centrifugal speed (Zimm et al. 1976, Blucher 1982) an alternative method is often applied, i .e. the neutral elution/retention assay (Bradley and Kohn 1979) . This approach, however, does not provide absolute measures of dsb frequencies, and requires thorough calibration for which some non-obvious assumptions have to be made (Hutchinson 1989) . Its applicability is therefore still controversial (Okayasu and Iliakis 1989, Radford 1990) . The situation is quite different with yeast cells. The small sizes of (native) DNA molecules and the (probably related) low cellular radiation sensitivity allows sedimentation analysis of dsb and cell survival measurement within the same dose-range (Frankenberg-Schwager et al . . 1979) . This has been extensively studied with sparsely ionizing radiation and the role of (unrepaired) DNA dsb as lethal lesions in yeast appears to be conclusive (Frankenberg et al . 1981) . For high-LET radiations and very heavy ions in particular a considerable amount of data has been accumulated on yeast cell killing (Manney et al. 1963, Bertsche 1978, Schopfer et al. 1982) but apart from experiments with a-particles (Frankenberg et al . 1981) or ultrasoft X-rays (Frankenberg et al. 1986) no information is available on dsb induction with densely ionizing radiations . In the present investigation dsb induction was measured by the sedimentation technique in yeast cells exposed to a variety of energetic heavy ions (provided by the UNILAC facility at the Gesellschaft fur Schwerionenforschung, GSI) . The derived dsb production cross-sections are compared with inactivationstudies performed in parallel for the same cells under identical irradiation conditions . Some of the data have already been presented in a preliminary communication (Akpa et al . 1991) .
2. Materials and methods 2.1
Cell growth and exposure conditions
Cells of a diploid strain of Saccharomyces cerevisiae (211 *B) auxotroph for 2'-deoxythymidine-5'-mono-
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T. C . Akpa et al . Table 1 . Physical parameters of the heavy ion beams, calculated DNA dsb production cross-section ad, b: normalized to 109 g mol -1 ) and derived inactivation cross-sections a ; (final slopes of the survival curves) . RBE& , is the relative biological effectiveness for the induction of double-strand breaks Heavy ions
Elm (MeV/u)
dE/dx (keV/µm)
adsb (10 -8 cm 2 )
ai (10 -8 cm 2 )
RBEd,ba
Xe Au Pb
4.9 9.8 14-4 5 .0 12-9 13 . 4 14 .0 11 . 8 9. 1 12 . 7
661 418 316 1 762 1020 994 966 6 427 11 649 11 125
0 . 64+0 . 11 0-58 + 0-09 0 . 41 + 0-08 0-96 ± 0 . 22 0 . 70+ _ 0 . 14 0-57 + 0 . 05 0 . 71+ _ 0 . 12 2 . 57+0 . 33 5-37 + 1-69 2-85 + 0-70
0 . 66+0 . 01 0-85 + 0-08 0 . 37 + 0 . 02 1-03 + 0 . 28 0 . 68+_0 . 01 0 . 54 + 0-03 0 . 55+_0 . 04 1 . 96+0 . 14 4 . 44 + 0 . 10 2-25 ± 0 . 30
1-04 1-49 1 . 39 0 . 59 0-74 0-62 0-76 0-43 0-49 0-28
ab
0 . 875
110
0-26 ± 0-02
Ne
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Ar
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'Relative effectiveness for dsb induction was calculated using the respective value for 30 MeV electrons of 5-8 x 10 -12 g -1 mol Gy-1 (Frankenberg et al. 1981) . 'Data for a-particles from Frankenberg et al . (1981) .
phosphate were grown overnight at 30 ° C in 4% glucose, 1 . 3% yeast nitrogen base (w/o amino acids), 04% casamino acids (w/o vitamins) and 7 pg/ml 5'dTMP (Sigma, Deisenhofen, Germany) . Following appropriate dilution they were inoculated in fresh medium to which 148 KBq/ml of methyl- 3 H-dTMP (Amersham-Buchler, Braunschweig, Germany) was added, and incubated for further 15 h during which the cell culture reached stationary growth phase (approximately 10 8 cells/ml) . Subsequently cells were collected by centrifugation, washed and resuspended in 67 mM phosphate buffer (pH 7 . 5) . At this stage labelled cells could be stored on ice (for up to 3 h) prior to the irradiation procedure . A total of 10' cells from the ice-cold suspension were collected as monolayers on membrane filters (3 . 5 cm diameter, 0 . 6 pm pore size; Sartorius, Gottingen, Germany) . For the exposure to heave ions a sample changer was used (Kraft et al. 1980) operating at room temperature, total exposure time for an experiment was always between approximately 20 and 40 min. Particle fluence was determined as described in detail earlier (Kraft et al. 1980) . The specific particle energies were calculated for the cell surface by using stopping-power data applied to the vacuum pipe exit window and the air gap between window and sample . Ion stopping-power values were calculated according to a procedure described by Kiefer (1987) . Heavy ion physical parameters are summarized in Table 1 .
2 .2. DNA dsh determination and survival assay Following exposure cells were thoroughly shaken off the filters by stirring them in phosphate buffer (pH 7) for 2 min and divided into two aliquots (5 x 10 6 cells each) that allowed the subsequent dsb determination to be run in duplicate . Cells were protoplasted as described elsewhere (FrankenbergSchwager et al . 1979) and resuspended in 0 . 1 M NaCl, 0 . 2 M EDTA, which caused them to collapse without liberating their DNA (Blamire et al. 1972) . For liberation of DNA, samples were layered on an equal volume of lysing layer on top of a neutral sucrose density gradient (5-20%) . To avoid speed effects the released DNA was sedimented for 21 h at 9000 r .p.m . in a Beckman ultracentrifuge (Model L5-50B) using the rotor SW 40 Ti . Gradients were fractionated onto glass fibre filters (Schleicher & Schiill) . The filters were dried and the highmolecular-mass DNA precipitated with ice-cold 6% trichloroacetic acid . The filters were then washed with ethanol and dried . A toluene-based scintillation liquid was added to each filter in a scintillation vial and the radioactivity was determined . The mean molar mass M (in g mol - 1) of the DNA in each fraction was calculated from the sedimentation coefficient s (in Svedberg units), using the empirical expression (Frankenberg-Schwager et al . 1979) s = 3 . 35 + 0 .013816
MO-445
(1)
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Heavy ion-induced DNA double-strand breaks in yeast
based on sedimentation of DNA from phages T2, T5 and T7 . The observed distribution in M was compared to theoretical distributions calculated as follows . An average number of random breaks per unit mass of DNA was assumed, and the expected distribution in mass was calculated by summing the distributions for each chromosome (of known mass) . The process was repeated for other assumed numbers of breaks, and the number of breaks induced by the radiation' treatment was taken to be that which produced the distribution fitting the observed most closely, as determined by a leastsquares procedure . This analysis demands that the dsb yield function has a zero ordinate intercept . Cell survival was measured with unlabelled cells from the same original culture (see §2 .1) . Immediately following exposure cells were taken off the filters as above and subjected to a standard colony formation assay . The growth medium contained 2% glucose, 0 .5% yeast extract and 7µg/ml 5'-dTMP . 3. Results Figures 1 a-c show the data on DNA dsb induction obtained with yeast cells of strain 211 *B after exposure to accelerated charged particles . They are given as mean number of dsb (N) per unit molar mass (mr ) of DNA . A linear dependence of the N/m r values on particle fluence is found in all cases . The slopes of the induction curves were obtained by linear regression . Data from multiple measurements were weighted accordingly. For Au ions the value at the highest exposure (see Figure 1) was omitted from the analysis . The slopes were normalized to 10 9 g mol -1 and are listed as 'dsb production cross-sections' a dsb in Table 1 . They are therefore relative values with respect to a DNA molecule of this size . The corresponding survival curves are depicted in Figures 2a-c . In most cases they have shoulders-a phenomenon typically found with diploid yeast cells even with very high LET radiations (Schopfer et al. 1982) . As a parameter of cellular sensitivity final slopes were derived by fitting the data to the multitarget formula (without any mechanistic implications) . They are given as inactivation cross-sections (a;, see Table 1) although it is recognized that this concept (Pollard et al. 1952) would strictly apply to a single-hit type response only and may not necessarily be inferred for heavy ion induced yeast cell killing (Kiefer 1985) . Attempts to fit the data to the linearquadratic survival expression were found to be inappropriate . The magnitude of the shoulder varies considerably for the different particle radiations (no common extrapolation numbers) and a correlation
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with radiation quality could not be found . A further analysis with respect to this parameter is therefore not possible .
4. Discussion The fluence dependencies for the induction of DNA dsb in yeast cells from very heavy ion irradiation are generally linear, as was also found with 30 MeV electron or 3 . 5 MeV a-particle exposure (using the same cell strain : Frankenberg et al . 1981) . In Figure 3 derived dsb production cross-sections are plotted versus unrestricted linear energy transfer (LEToo) of the particle radiations, the value for aparticles (see Table 1) is included . Also drawn is a a versus LET curve (dotted line) calculated for a relative biological effectiveness (RBE) of unity with regard to the dsb yield for 30 MeV electrons of 5 .8 x 10 -12 g -1 mol Gy - ' (and normalized to 10 9 g mol') . Although the presently collected yeast dsb data are not sufficient to allow a complete description of this high LET radiation action they confirm a general trend reported earlier for induction in mammalian cells when the neutral sedimentation technique was also employed (Kampf and Eichhorn 1983, Blucher 1988) and extend such measurements to very heavy ions. In order to facilitate a comparison these published yields were normalized to 10 9 g mol - ' and-because mostly given in units of dosetransformed to units in fluence . Figure 3 shows our yeast dsb data from Table 1, together with the mammalian cell results . The data by Ritter et al. . (1977)-frequently quoted as DNA `double-strand breakage'-are not included because they refer to non-rejoined total strand breaks following a repair incubation . The presumption that this value reflects predominantly dsb induction is not justified per se. A comparison with the mammalian cell data by Kampf and Eichhorn (1983) appears particularly interesting because the respective dsb yield for sparsely ionizing radiation (X-rays : 5.3 x 10 -12 g -1 mol Gy - ' with cells exposed as monolayers) is close to the yeast cell value given above (Frankenberg et al . 1981) . For LET values where the two data sets -1 ) overlap (around 300-400 keV µm also quite similar dsb yields are found per unit molar mass of yeast DNA and mammalian DNA which is in line with results on a-particle induced dsb (Frankenberg et al. 1981, Kampf and Eichhorn 1983 ; included in Figure 3) . Our yeast results therefore add to available data sets and some features of the LETdependent dsb induction may be considered :
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(1) It appears that the dsb production cross-sections `level off at LET values above about 10 2 keV µm -1 with a plateau region up to 103 keV µm -1 . This indicates a finite probability (or a saturation) to produce dsb by a single particle
traversal as long as the action by far-reaching delta electrons in the track is negligible (see : Kiefer 1985 for a review) . It has been shown that already for this `plateau region' inactivation cross-sections (for yeast and mammalian
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Heavy ion-induced DNA double-strand breaks in yeast
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cells) depend both on LET and particle velocity yielding separate curves for different types of heavy ions . As can be seen from Table 1 the particle-specific energies (Elm) that govern possible delta electron effects in the so-called `penumbra' are in a relatively narrow range .
But the plateau could be partly due to a `shrinkage' of the penumbra for a given ion type as LET increases (decrease of Elm) . The same may be true for the measurements by Kampf and Eichhorn (1983) where the plateau region is seen with charged particles of low velocity (1 .05
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T. C. Akpa et al . MeV/u and 0 .45 MeV/u a-particles, 6 . 7 MeV/u carbon ions : energies for these ions given in Tolkendorf and Eichhorn 1983) . The influence of track structure becomes increasingly relevant when the number of secondary electrons produced per particle increases which is proportional to Z* 2 /fl of the ion (Z* is the effective charge and the ion velocity relative to that of light in vacuo : Bethe 1930) . Accordingly, the effectiveness per particle (cross-section) to produce damage (e.g. DNA double-strand breaks) should be expected to rise above the `plateau value' for ions of higher values of Z* 2 /fl2 (which is also roughly proportional to LET for the narrow energy range of ions studied here) . This expectation is borne out in the present investigation (Figure 3) . According to current track structure models (e.g. Butts and Katz 1967, Katz 1988, Kiefer 1983) cross-sections depend on particle velocity at least for very heavy ions . This was qualitatively verified in a number of experimental studies (summarized in Kiefer 1985, Kraft 1987) including measurements on (total) strand break induction in cells (Rydberg 1985, Aufderheide et al . 1987) or dsb production in dilute aqueous DNA solution without added protective agents (Roots et al . 1990) . But no information on dsb induction in cells or in an in
vitro model system aimed to simulate the intracellular environment is available from the literature that demonstrate the postulated dependence on particle velocity . The data obtained with yeast cells do also not yet provide sufficient information with respect to this LET range . (2) Another important aspect is the dependence of relative biological effectiveness (RBE) for dsb induction on radiation quality-i .e. its possible increase in the LET range below the `plateau' region . This can be more clearly seen in a plot of dsb yields per unit dose versus LET which is shown in Figure 4 . Such a representation also reflects the different sensitivities to sparsely ionizing radiations with different experimental systems which are used as a base-line . The yeast results also indicate that RBE values rise above unity (compare also with Figure 3 : data points above the dotted line) . Whether a relative effectiveness of 4-5 as seen with mammalian cells (Kampf and Eichhorn 1983) can also be found in yeast remains to be resolved, and further measurements are obviously needed in the LET range below approximately 300 keV pm -t . It should be mentioned that several studies using the neutral elution technique showed RBE values for initial dsb in mammalian cells of about unity (neutrons, a-particles : Prise et al .
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LET / keV Pm _1 Figure 3 . Double-strand break production cross-sections for yeast (from Table 1) plotted versus heavy ion LET (circles) . The squared symbol is for a-particles . The dotted line is drawn for a relative biological effectiveness of unity with regard to the dsb yield for 30 MeV electrons in yeast (see text) . Also shown are the mammalian cell data by Kampf and Eichhorn (1983 ; triangles) and Blucher (1988 ; diamond) .
Heavy ion-induced DNA double-strand breaks in yeast 6
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1987, Peak et al. 1991) . The apparent discrepancy between the two experimental approaches awaits clarification . DNA dsb are thought to be the main lesions responsible for cell inactivation by ionizing radiations, although the evidence is still to a great extent indirect (see FrankenbergSchwager 1990 for review and discussion) . In the present case there is obviously no direct proportionality between the number of induced dsb and lethality since dsb induction is always linear with fluence (or dose) while the survival curves have shoulders in many cases . It is important to note that both endpoints were measured in the same fluence range, a situation which is different when mammalian cells are used . There is, however, a very good correlation (in fact a proportionality) between the inactivation cross-section derived from the final slopes of the yeast survival curves and dsb induction crosssection over the large LET-range studied (Figure 5) . From regression analysis of these data (linear dependence, correlation coefficient of 0 . 99) a value of 1 . 20 ± 0 . 96 for the number of induced dsb per 10 9 g mol -1 and per lethal event is obtained . Using the DNA content of a diploid yeast cell (1 .8 ± 0 . 4) x 10 10 g mol - ' : Lauer et al . 1977) one may calculate that about 22 DNA dsb are induced per cell at the mean lethal exposure-a value that is nearly identical
to the corresponding number for 30 meV electrons (Do for survival of 208 Gy : FrankenbergSchwager et al . 1979) . It is a generally accepted view that cell killing relates to unrepaired or misrepaired dsb rather than induced dsb . Also for heavy ion effects on yeast it was shown that the expression of lethality depends on dsb repair (Schopfer et al . 1982, Kiefer and Schneider 1991) . From the results depicted in Figure 5 it can be concluded that damage processing in diploid yeast is similarly effective for sparsely ionizing radiation and the different heavy ions used here if the survival curve's terminal part is considered . This is obviously different for low exposures or when fixed survival levels are compared : the extrapolation numbers derived from very heavy ion survival curves (for diploid yeast see also Schopfer et al . 1982) were consistently smaller than that found with sparsely ionizing radiation. This is taken to indicate that it is not a different class of less reparable ('high-LET type') dsb that play a major role in heavy particle yeast cell killing but that repair effectiveness is reduced due to the close proximity of primary lesions as is seen with low-LET radiation at high exposure . It needs to be . emphasized that these considerations are limited to yeast . No such relationship between dsb induction and cell killing when high- and low-LET radiations are compared is seen for mammalian cells (e.g. Prise
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10.0
1.0
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OSB production cross-section / 10-8 cm2 Figure 5 . Inactivation cross-sections derived from the final slopes of the heavy ion survival curves plotted versus the respective dsb production cross-sections (data pairs from Table 1) .
1987) . This important difference between yeast and mammalian cells remains to be resolved. et al.
Acknowledgements The authors thank Beate Barth for excellent technical assistance . This work was partially supported by the Gesellschaft ftir Schwerionenforschung (GSI), Darmstadt, Germany .
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J . E . and TOBIAS, C . A . 1977, HighLET radiations induce a large proportion of nonrejoining DNA breaks . Nature, 266, 653-655 . ROOTS, R ., YANG, T . C., CRAISE, L ., BLAKELY, E . A. and RITTER, M. A., CLEAVER,
TOBIAS, C . A ., 1979, Impaired repair capacity of DNA breaks induced in mammalian cellular DNA by accelerated heavy ions . Radiation Research, 78, 38-49 . ROOTS, R ., HOLLEY, W., CHATTERJEE, A ., IRIZARRY, M . and KRAFT, G, 1990, The formation of strand breaks in DNA after high-LET irradiation : a comparison of data from in vitro and cellular systems . International Journal of Radiation Biology, 58, 55-69 . RYDBERG, B ., 1985, DNA strand breaks induced by low-energy heavy ions. International Journal of Radiation Biology, 47, 57-61 . SCHOPFER, F., SCHNEIDER, E ., RASE, S ., KIEFER, J . and KRAFT, G., 1982, Heavy-ion effects on yeast: survival and recovery in vegetative cells of different sensitivities . Radiation Research, 92, 30-46. TODD, P. W . and TOBIAS, C . A ., 1974, Cellular radiation biology . In : Space Radiation Biology and Related Topics . Edited by: C . A. Tobias and P. W . Todd, pp. 142-187 (Academic Press, New York) . TOLKENDORF, E. and EICHHORN, K ., 1983, Effect of ionizing radiation of different linear energy transfer on the induction of cellular death and of chromosome aberrations in cells of the Chinese hamster. Studia Biophysica, 95, 43-56. ZIMM, B . H ., SCHUMAKER, V. N . and ZIMM, C . B., 1976, Anomalies in sedimentation . V. Chains at high fields, practical consequences . Biophysical Chemistry, 5, 265-270.
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RADIAT . BIOL .,
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Heavy ion-induced DNA double-strand breaks in yeast T. C. AKPAf , K . J. WEBERt§, E . SCHNEIDERt, J . KIEFERt*, M. FRANKENBERG-SCHWAGER¶, R . HARBICH¶ and D . FRANKENBERG¶
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(Received 9 August 1991 ; second revision received 30 March 1992 ; accepted 12 April 1992)
Abstract. DNA double-strand break (dsb) induction in diploid yeast was measured by neutral sucrose sedimentation after exposure to very heavy ions with values of linear energy transfer . Linear (LET) ranging from about 300 to 11500 keV/pm fluence dependencies were found in all cases from which dsb production cross-sections (ad .b ) could be calculated . Corresponding cross-sections for cell killing ((a ;) were derived from final slopes of survival curves measured in parallel and for the same fluence range. A close correlation was found between a ; and a d,b . It is calculated that over the entire LET range, including 30 Me V electron irradiation, about 22 dsb are induced per lethal event when high exposures are considered .
1. Introduction DNA double-strand breaks (dsb) appear to be the most important DNA lesions induced by ionizing radiations related to chromosomal aberrations and cell death (see Frankenberg-Schwager 1990 for a recent summary) . This is normally implied from the close correlations between observed cellular radiation response and measured dsb frequencies - as reported for many different cell lines - and a parallelism in the change of these experimental endpoints when modifying conditions were employed . Accordingly, the influence of radiation quality (i.e. linear energy transfer, LET) on lesion induction and damage expression has found wide attention, but despite numerous studies a conclusive picture is proving to be hard to reach (Todd and Tobias 1974, Kiefer 1985, Kraft 1987, Roots et al . 1990) . The detection of DNA dsb in mammalian cells for a `biologically relevant' dose range (below approximately 20 Gy) is largely hampered by the high residual molecular weights of DNA fragments generated with such doses . Since they may not easily be *To whom correspondence should be addressed . tStrahlenzentrum der Justus-Liebig-Universitat, GieBen, Germany . ¶Institut fur Biophysikalische Strahenforschung der GSF, Frankfurt-Main, Germany. $Present address : Physics Department, Ahmadu Bello University, Zaria, Nigeria . §Present address : Strahlenklinik der Universitat, Heidelberg, Germany .
analysed by the conventional neutral sucrose sedimentation technique at low centrifugal speed (Zimm et al. 1976, Blucher 1982) an alternative method is often applied, i .e. the neutral elution/retention assay (Bradley and Kohn 1979) . This approach, however, does not provide absolute measures of dsb frequencies, and requires thorough calibration for which some non-obvious assumptions have to be made (Hutchinson 1989) . Its applicability is therefore still controversial (Okayasu and Iliakis 1989, Radford 1990) . The situation is quite different with yeast cells. The small sizes of (native) DNA molecules and the (probably related) low cellular radiation sensitivity allows sedimentation analysis of dsb and cell survival measurement within the same dose-range (Frankenberg-Schwager et al . . 1979) . This has been extensively studied with sparsely ionizing radiation and the role of (unrepaired) DNA dsb as lethal lesions in yeast appears to be conclusive (Frankenberg et al . 1981) . For high-LET radiations and very heavy ions in particular a considerable amount of data has been accumulated on yeast cell killing (Manney et al. 1963, Bertsche 1978, Schopfer et al. 1982) but apart from experiments with a-particles (Frankenberg et al . 1981) or ultrasoft X-rays (Frankenberg et al. 1986) no information is available on dsb induction with densely ionizing radiations . In the present investigation dsb induction was measured by the sedimentation technique in yeast cells exposed to a variety of energetic heavy ions (provided by the UNILAC facility at the Gesellschaft fur Schwerionenforschung, GSI) . The derived dsb production cross-sections are compared with inactivationstudies performed in parallel for the same cells under identical irradiation conditions . Some of the data have already been presented in a preliminary communication (Akpa et al . 1991) .
2. Materials and methods 2.1
Cell growth and exposure conditions
Cells of a diploid strain of Saccharomyces cerevisiae (211 *B) auxotroph for 2'-deoxythymidine-5'-mono-
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T. C . Akpa et al . Table 1 . Physical parameters of the heavy ion beams, calculated DNA dsb production cross-section ad, b: normalized to 109 g mol -1 ) and derived inactivation cross-sections a ; (final slopes of the survival curves) . RBE& , is the relative biological effectiveness for the induction of double-strand breaks Heavy ions
Elm (MeV/u)
dE/dx (keV/µm)
adsb (10 -8 cm 2 )
ai (10 -8 cm 2 )
RBEd,ba
Xe Au Pb
4.9 9.8 14-4 5 .0 12-9 13 . 4 14 .0 11 . 8 9. 1 12 . 7
661 418 316 1 762 1020 994 966 6 427 11 649 11 125
0 . 64+0 . 11 0-58 + 0-09 0 . 41 + 0-08 0-96 ± 0 . 22 0 . 70+ _ 0 . 14 0-57 + 0 . 05 0 . 71+ _ 0 . 12 2 . 57+0 . 33 5-37 + 1-69 2-85 + 0-70
0 . 66+0 . 01 0-85 + 0-08 0 . 37 + 0 . 02 1-03 + 0 . 28 0 . 68+_0 . 01 0 . 54 + 0-03 0 . 55+_0 . 04 1 . 96+0 . 14 4 . 44 + 0 . 10 2-25 ± 0 . 30
1-04 1-49 1 . 39 0 . 59 0-74 0-62 0-76 0-43 0-49 0-28
ab
0 . 875
110
0-26 ± 0-02
Ne
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Ar
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'Relative effectiveness for dsb induction was calculated using the respective value for 30 MeV electrons of 5-8 x 10 -12 g -1 mol Gy-1 (Frankenberg et al. 1981) . 'Data for a-particles from Frankenberg et al . (1981) .
phosphate were grown overnight at 30 ° C in 4% glucose, 1 . 3% yeast nitrogen base (w/o amino acids), 04% casamino acids (w/o vitamins) and 7 pg/ml 5'dTMP (Sigma, Deisenhofen, Germany) . Following appropriate dilution they were inoculated in fresh medium to which 148 KBq/ml of methyl- 3 H-dTMP (Amersham-Buchler, Braunschweig, Germany) was added, and incubated for further 15 h during which the cell culture reached stationary growth phase (approximately 10 8 cells/ml) . Subsequently cells were collected by centrifugation, washed and resuspended in 67 mM phosphate buffer (pH 7 . 5) . At this stage labelled cells could be stored on ice (for up to 3 h) prior to the irradiation procedure . A total of 10' cells from the ice-cold suspension were collected as monolayers on membrane filters (3 . 5 cm diameter, 0 . 6 pm pore size; Sartorius, Gottingen, Germany) . For the exposure to heave ions a sample changer was used (Kraft et al. 1980) operating at room temperature, total exposure time for an experiment was always between approximately 20 and 40 min. Particle fluence was determined as described in detail earlier (Kraft et al. 1980) . The specific particle energies were calculated for the cell surface by using stopping-power data applied to the vacuum pipe exit window and the air gap between window and sample . Ion stopping-power values were calculated according to a procedure described by Kiefer (1987) . Heavy ion physical parameters are summarized in Table 1 .
2 .2. DNA dsh determination and survival assay Following exposure cells were thoroughly shaken off the filters by stirring them in phosphate buffer (pH 7) for 2 min and divided into two aliquots (5 x 10 6 cells each) that allowed the subsequent dsb determination to be run in duplicate . Cells were protoplasted as described elsewhere (FrankenbergSchwager et al . 1979) and resuspended in 0 . 1 M NaCl, 0 . 2 M EDTA, which caused them to collapse without liberating their DNA (Blamire et al. 1972) . For liberation of DNA, samples were layered on an equal volume of lysing layer on top of a neutral sucrose density gradient (5-20%) . To avoid speed effects the released DNA was sedimented for 21 h at 9000 r .p.m . in a Beckman ultracentrifuge (Model L5-50B) using the rotor SW 40 Ti . Gradients were fractionated onto glass fibre filters (Schleicher & Schiill) . The filters were dried and the highmolecular-mass DNA precipitated with ice-cold 6% trichloroacetic acid . The filters were then washed with ethanol and dried . A toluene-based scintillation liquid was added to each filter in a scintillation vial and the radioactivity was determined . The mean molar mass M (in g mol - 1) of the DNA in each fraction was calculated from the sedimentation coefficient s (in Svedberg units), using the empirical expression (Frankenberg-Schwager et al . 1979) s = 3 . 35 + 0 .013816
MO-445
(1)
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Heavy ion-induced DNA double-strand breaks in yeast
based on sedimentation of DNA from phages T2, T5 and T7 . The observed distribution in M was compared to theoretical distributions calculated as follows . An average number of random breaks per unit mass of DNA was assumed, and the expected distribution in mass was calculated by summing the distributions for each chromosome (of known mass) . The process was repeated for other assumed numbers of breaks, and the number of breaks induced by the radiation' treatment was taken to be that which produced the distribution fitting the observed most closely, as determined by a leastsquares procedure . This analysis demands that the dsb yield function has a zero ordinate intercept . Cell survival was measured with unlabelled cells from the same original culture (see §2 .1) . Immediately following exposure cells were taken off the filters as above and subjected to a standard colony formation assay . The growth medium contained 2% glucose, 0 .5% yeast extract and 7µg/ml 5'-dTMP . 3. Results Figures 1 a-c show the data on DNA dsb induction obtained with yeast cells of strain 211 *B after exposure to accelerated charged particles . They are given as mean number of dsb (N) per unit molar mass (mr ) of DNA . A linear dependence of the N/m r values on particle fluence is found in all cases . The slopes of the induction curves were obtained by linear regression . Data from multiple measurements were weighted accordingly. For Au ions the value at the highest exposure (see Figure 1) was omitted from the analysis . The slopes were normalized to 10 9 g mol -1 and are listed as 'dsb production cross-sections' a dsb in Table 1 . They are therefore relative values with respect to a DNA molecule of this size . The corresponding survival curves are depicted in Figures 2a-c . In most cases they have shoulders-a phenomenon typically found with diploid yeast cells even with very high LET radiations (Schopfer et al. 1982) . As a parameter of cellular sensitivity final slopes were derived by fitting the data to the multitarget formula (without any mechanistic implications) . They are given as inactivation cross-sections (a;, see Table 1) although it is recognized that this concept (Pollard et al. 1952) would strictly apply to a single-hit type response only and may not necessarily be inferred for heavy ion induced yeast cell killing (Kiefer 1985) . Attempts to fit the data to the linearquadratic survival expression were found to be inappropriate . The magnitude of the shoulder varies considerably for the different particle radiations (no common extrapolation numbers) and a correlation
281
with radiation quality could not be found . A further analysis with respect to this parameter is therefore not possible .
4. Discussion The fluence dependencies for the induction of DNA dsb in yeast cells from very heavy ion irradiation are generally linear, as was also found with 30 MeV electron or 3 . 5 MeV a-particle exposure (using the same cell strain : Frankenberg et al . 1981) . In Figure 3 derived dsb production cross-sections are plotted versus unrestricted linear energy transfer (LEToo) of the particle radiations, the value for aparticles (see Table 1) is included . Also drawn is a a versus LET curve (dotted line) calculated for a relative biological effectiveness (RBE) of unity with regard to the dsb yield for 30 MeV electrons of 5 .8 x 10 -12 g -1 mol Gy - ' (and normalized to 10 9 g mol') . Although the presently collected yeast dsb data are not sufficient to allow a complete description of this high LET radiation action they confirm a general trend reported earlier for induction in mammalian cells when the neutral sedimentation technique was also employed (Kampf and Eichhorn 1983, Blucher 1988) and extend such measurements to very heavy ions. In order to facilitate a comparison these published yields were normalized to 10 9 g mol - ' and-because mostly given in units of dosetransformed to units in fluence . Figure 3 shows our yeast dsb data from Table 1, together with the mammalian cell results . The data by Ritter et al. . (1977)-frequently quoted as DNA `double-strand breakage'-are not included because they refer to non-rejoined total strand breaks following a repair incubation . The presumption that this value reflects predominantly dsb induction is not justified per se. A comparison with the mammalian cell data by Kampf and Eichhorn (1983) appears particularly interesting because the respective dsb yield for sparsely ionizing radiation (X-rays : 5.3 x 10 -12 g -1 mol Gy - ' with cells exposed as monolayers) is close to the yeast cell value given above (Frankenberg et al . 1981) . For LET values where the two data sets -1 ) overlap (around 300-400 keV µm also quite similar dsb yields are found per unit molar mass of yeast DNA and mammalian DNA which is in line with results on a-particle induced dsb (Frankenberg et al. 1981, Kampf and Eichhorn 1983 ; included in Figure 3) . Our yeast results therefore add to available data sets and some features of the LETdependent dsb induction may be considered :
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(1) It appears that the dsb production cross-sections `level off at LET values above about 10 2 keV µm -1 with a plateau region up to 103 keV µm -1 . This indicates a finite probability (or a saturation) to produce dsb by a single particle
traversal as long as the action by far-reaching delta electrons in the track is negligible (see : Kiefer 1985 for a review) . It has been shown that already for this `plateau region' inactivation cross-sections (for yeast and mammalian
20
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Heavy ion-induced DNA double-strand breaks in yeast
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cells) depend both on LET and particle velocity yielding separate curves for different types of heavy ions . As can be seen from Table 1 the particle-specific energies (Elm) that govern possible delta electron effects in the so-called `penumbra' are in a relatively narrow range .
But the plateau could be partly due to a `shrinkage' of the penumbra for a given ion type as LET increases (decrease of Elm) . The same may be true for the measurements by Kampf and Eichhorn (1983) where the plateau region is seen with charged particles of low velocity (1 .05
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T. C. Akpa et al . MeV/u and 0 .45 MeV/u a-particles, 6 . 7 MeV/u carbon ions : energies for these ions given in Tolkendorf and Eichhorn 1983) . The influence of track structure becomes increasingly relevant when the number of secondary electrons produced per particle increases which is proportional to Z* 2 /fl of the ion (Z* is the effective charge and the ion velocity relative to that of light in vacuo : Bethe 1930) . Accordingly, the effectiveness per particle (cross-section) to produce damage (e.g. DNA double-strand breaks) should be expected to rise above the `plateau value' for ions of higher values of Z* 2 /fl2 (which is also roughly proportional to LET for the narrow energy range of ions studied here) . This expectation is borne out in the present investigation (Figure 3) . According to current track structure models (e.g. Butts and Katz 1967, Katz 1988, Kiefer 1983) cross-sections depend on particle velocity at least for very heavy ions . This was qualitatively verified in a number of experimental studies (summarized in Kiefer 1985, Kraft 1987) including measurements on (total) strand break induction in cells (Rydberg 1985, Aufderheide et al . 1987) or dsb production in dilute aqueous DNA solution without added protective agents (Roots et al . 1990) . But no information on dsb induction in cells or in an in
vitro model system aimed to simulate the intracellular environment is available from the literature that demonstrate the postulated dependence on particle velocity . The data obtained with yeast cells do also not yet provide sufficient information with respect to this LET range . (2) Another important aspect is the dependence of relative biological effectiveness (RBE) for dsb induction on radiation quality-i .e. its possible increase in the LET range below the `plateau' region . This can be more clearly seen in a plot of dsb yields per unit dose versus LET which is shown in Figure 4 . Such a representation also reflects the different sensitivities to sparsely ionizing radiations with different experimental systems which are used as a base-line . The yeast results also indicate that RBE values rise above unity (compare also with Figure 3 : data points above the dotted line) . Whether a relative effectiveness of 4-5 as seen with mammalian cells (Kampf and Eichhorn 1983) can also be found in yeast remains to be resolved, and further measurements are obviously needed in the LET range below approximately 300 keV pm -t . It should be mentioned that several studies using the neutral elution technique showed RBE values for initial dsb in mammalian cells of about unity (neutrons, a-particles : Prise et al .
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Heavy ion-induced DNA double-strand breaks in yeast 6
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1987, Peak et al. 1991) . The apparent discrepancy between the two experimental approaches awaits clarification . DNA dsb are thought to be the main lesions responsible for cell inactivation by ionizing radiations, although the evidence is still to a great extent indirect (see FrankenbergSchwager 1990 for review and discussion) . In the present case there is obviously no direct proportionality between the number of induced dsb and lethality since dsb induction is always linear with fluence (or dose) while the survival curves have shoulders in many cases . It is important to note that both endpoints were measured in the same fluence range, a situation which is different when mammalian cells are used . There is, however, a very good correlation (in fact a proportionality) between the inactivation cross-section derived from the final slopes of the yeast survival curves and dsb induction crosssection over the large LET-range studied (Figure 5) . From regression analysis of these data (linear dependence, correlation coefficient of 0 . 99) a value of 1 . 20 ± 0 . 96 for the number of induced dsb per 10 9 g mol -1 and per lethal event is obtained . Using the DNA content of a diploid yeast cell (1 .8 ± 0 . 4) x 10 10 g mol - ' : Lauer et al . 1977) one may calculate that about 22 DNA dsb are induced per cell at the mean lethal exposure-a value that is nearly identical
to the corresponding number for 30 meV electrons (Do for survival of 208 Gy : FrankenbergSchwager et al . 1979) . It is a generally accepted view that cell killing relates to unrepaired or misrepaired dsb rather than induced dsb . Also for heavy ion effects on yeast it was shown that the expression of lethality depends on dsb repair (Schopfer et al . 1982, Kiefer and Schneider 1991) . From the results depicted in Figure 5 it can be concluded that damage processing in diploid yeast is similarly effective for sparsely ionizing radiation and the different heavy ions used here if the survival curve's terminal part is considered . This is obviously different for low exposures or when fixed survival levels are compared : the extrapolation numbers derived from very heavy ion survival curves (for diploid yeast see also Schopfer et al . 1982) were consistently smaller than that found with sparsely ionizing radiation. This is taken to indicate that it is not a different class of less reparable ('high-LET type') dsb that play a major role in heavy particle yeast cell killing but that repair effectiveness is reduced due to the close proximity of primary lesions as is seen with low-LET radiation at high exposure . It needs to be . emphasized that these considerations are limited to yeast . No such relationship between dsb induction and cell killing when high- and low-LET radiations are compared is seen for mammalian cells (e.g. Prise
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10.0
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OSB production cross-section / 10-8 cm2 Figure 5 . Inactivation cross-sections derived from the final slopes of the heavy ion survival curves plotted versus the respective dsb production cross-sections (data pairs from Table 1) .
1987) . This important difference between yeast and mammalian cells remains to be resolved. et al.
Acknowledgements The authors thank Beate Barth for excellent technical assistance . This work was partially supported by the Gesellschaft ftir Schwerionenforschung (GSI), Darmstadt, Germany .
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