¹Groupe de Physique des Solides, Universités Paris 6 et Paris 7, Paris, France; ²Laboratoire de Radiobiologie et Oncologie, Fontenay aux Roses, France; ³Laboratoire de Génotoxicité et
Modulation de l’Expression Génique, Institut Curie Recherche, Orsay, France
Address all correspondence to Annie Chetioui, Groupe de Physique des Solides, Universites Paris 6 et
7, CNRS UMR 75-88, 2 Place Jussieu, 75251 Paris Cedex 05, France; [email protected]
It has been proposed that unrepaired or misrepaired complex lesions of DNA are responsible for cell inactivation and chromosomal aberrations. Th e detailed features of the critical damage and the nature of initiating physical events are actively investigated. Here the role of inner-shell (core) ionizations in DNA atoms is studied. Ultrasoft X-rays from LURE synchrotron radiation have been used to mimic core events induced by ionizing radiations. For biological matter, inner-shell photoionization is indeed the main interaction channel of these radiations. Moreover, by tuning the X-ray energy below and above the carbon K-threshold, it is possible to achieve a two-fold increase in the number of core-ionizations in DNA for a same dose. Cell survival and chromosome aberrations have thus been studied at three iso-attenuated energies: 250, 350, and 810 eV. Relative biological effi ciencies (RBEs) for cell inactivation and chromosome aberrations were found to be strongly correlated with the yields of core events in DNA.
FIGURE 1. Scheme of outer-shell and inner-shell ionizations by charged particles. Energies indicated for secondary
electrons are typical mean energies for particles considered in radiobiological studies.
INACTIVATION AND CHROMOSOMAL ABERRATIONS
JEPTO 2004, Volume 23, Number 1 83
chiefl y lead to the ejection of low-energy electrons.
Th eir mean kinetic energies are on the order of a
few tens of electron volts (eV), depending upon the
velocity of the incident particle.⁹¹⁰ On the contrary,
inner-shell ionizations have a low probability of oc-
currence (~ some ‰) but release two highly ionizing
electrons—secondary and Auger—with mean ener-
gies in the range of some hundreds eV. In addition,
multiple ionizations (more than two ionizations in
a fraction of cases) are induced in situ by single or
double Auger relaxation.¹¹
When created on the DNA, core events presum-
ably may lead to highly complex lesions, especially
when the two emitted electrons overlap the DNA
(Fıg. 2). Early experiments indeed demonstrated the
lethal and mutagenic eff ectiveness of core ionizations
K
Auger
electron
Secondary
electron
DNA
FIGURE 2. Proposed scheme for the induction of complex DNA double-strand breaks by core-ionization of DNA
atoms.
84 JEPTO 2004, Volume 23, Number 1
ARNAUD BOISSIÈRE ET AL.
H HeFNeArFeKr U
LET (keV/µm)
0.01
0.1
1
10
100
10 100 1000 10000
mammal
yeast
bacteria
Inac
tiva
tion
cro
ss s
ecti
ons
(µm
2 )
LET(keV/µm)
Inac
tiva
tion
cro
ssse
ctio
ns
(µ
m2 )
bact
yeast
mammal
FeAr
FAr
U
XeNi
U+PbXe
Kr
Ar U
PbXeKrAr
10 100 1000 10000
0.01
0.1
1
10
100
H
He
FIGURE 3. Correlation between cross-sections for cell inactivation by heavy ions15 and for the production of effi cient
core ionizations in DNA atoms.14
INACTIVATION AND CHROMOSOMAL ABERRATIONS
JEPTO 2004, Volume 23, Number 1 85
in the phosphorus (P) atoms of DNA.¹²¹³ However,
because of the low presence of these atoms in DNA
and their small ionization cross-sections by charged
particles compared to those of other DNA constitu-
ent atoms, P core ionizations are expected to have an
overall small contribution to radiation eff ects. On the
other hand, because of their much larger frequency,
inner-shell ionizations in carbon (C)-, nitrogen (N)-,
and oxygen (O)- atoms of the DNA might have a
major biological role. Th is was particularly suggested
by the correlation observed for heavy ions between
cell inactivation cross- sections and C, N, O inner-
shell ionization cross-sections (Fıg. 3).¹⁴
Materials and Methods
Simulation of Core Events Using
Ultrasoft X-Rays
Th e eff ect of core ionizations cannot be selectively
investigated using usually charged particles—fast
electrons (either as direct particles or secondary to
γ-rays), and/or ions—because those produce core
events with a very low probability. On the contrary,
for most ultrasoft X-rays, the inner-shell ioniza-
tion is the dominant channel of interaction with
biological matter (Table 1). Moreover, by adjusting
the X-ray energy above an atomic K threshold, it is
possible to set the energy of the photoelectron and
to mimic, up to a certain extent, the core events
created by electrons or ions. Fınally, by selecting a
given K-threshold among those of the C, N, and
O, one may target preferentially a specifi c atomic
species (Fıg. 4). Th is may allow the preferential ex-
citation of a given cell compartment. For instance,
because of the DNA richness in carbon atoms, by
using X-rays above the C K-threshold (290 eV) and
below the O K-threshold (540 eV), it is possible to
preferentially excite the DNA. For a same dose/cell,
the number of core events on DNA is thus about
two times larger for X-ray energies between 290
and 540 eV than for energies just below 290 eV or
just above 540 eV.
Table 1. Fractions and Schemes of Various Photoionization Channels of Ultrasoft X-Rays Interacting with
Partially Hydrated DNA* *12 water molecules per nucleotide
250 eV 380 eV 760 eV
Outer-shellionization
Inner-shellionization
H, C, N,O, Na, P
230 eV
H, C, N,O, Na, P
740 eV
H, C, N,O, Na, P
360 eV
6%70% 26%
O220 eV
500 eV
C90 eV
260 eV
P110 eV
115 eV
68%61%30%
P (240 eV ; 115 eV) 13% C (470 eV ; 260 eV) 14%
N (370 eV; 360 eV ) 9%
P (620 eV ; 115 eV) 3%
X-ray
Energyionization
86 JEPTO 2004, Volume 23, Number 1
ARNAUD BOISSIÈRE ET AL.
Experimental Setup and Physical Methods for
Biological Studies at Synchrotron Radiation
Facilities
Experimental Device
Experiments designed to mimic core events require
the use of tuneable monochromatic X-rays, which
can be obtained only at synchrotron radiation facili-
ties. Th e present studies have been performed in the
LURE laboratory at Orsay.
Th e experimental setup has been described else-
where.¹⁶ Because of the strong absorption of ul-
trasoft X-rays by matter, it was important to reduce
as much as possible the thickness of all materials
placed ahead of the biological samples, which have to
be kept at atmospheric pressure. In order to extract
the photon beam, a three-stage diff erential pump-
ing connects a very thin (150 nm) silicon nitride
window to the beam line (~10–⁹ Torr). Th e culture
fl asks are equipped with a very thin (~1 µm) 1 cm²
mylar basement through which cells are irradiated.
Th e Mylar thicknesses have been measured with an
accuracy of ~3% using 1 MeV α-particle energy loss
at the van de Graaff accelerator of GPS at Paris 6-7
University. Th e biological samples were positioned
on an X-Y translator in order to obtain uniform ir-
radiation of the cells by the 1 mm² beam.
Dosimetry
Doses have been calculated from particle fl uences.
Th e X-ray intensity was monitored with a silicon
photodiode at the end of each translation cycle.
Th e diode was calibrated by comparison with
an extrapolation ionization chamber. Because of
the strong X-ray absorption inside the biological
medium, the eff ectiveness of various soft X-rays
was compared at energies for which isoattenua-
tion was achieved inside the cell—namely, 250,
350, and 810 eV (Fıg. 4). For these X-rays, a 0.15
ratio between the mean nuclear dose and the en-
trance dose was calculated, using the mean values
of cytoplasm and nucleus thicknesses measured by
confocal microscopy. Experiments were performed
for entrance dose rates ~3 Gy min–¹—i.e., mean
nuclear dose rates of ~0.5 Gy min–¹. Uncertainties
on dose delivered at a given energy are about 20%.
Uncertainties on the ratio of doses delivered at two
diff erent energies are ~22%.
Biological Methods
Cell Growth and Flattening
Studies of cell survival were performed using Chi-
nese hamster V79 cells. Th e cells were cultured in
DMEM supplied with fetal calf serum (FCS) and
antibiotics. Incubator settings were 37 °C and 5%
CO₂. Two days before exposure, cells were plated on
1 cm² Mylar glued across a square aperture cut in
one face of a 25-cm³ Costar fl ask. Cells were seeded
with a density of 5 × 10⁴ cells/cm² in DMEM/10%
FCS. Twelve hours later, the medium was removed
and DMEM/3% FCS was added to limit cell pro-
liferation and keep most of the cells in G₀ phase.
However, some cells were still dividing, as seen by
optical and confocal microscopy. Irradiations were
performed at room temperature 48 hours after seed-
ing to let cells fl atten on Mylar.
Cell Survival
After irradiation, cells were incubated for 2 hours
before replating. Th ey were trypsinized and counted,
then seeded at low density: three dilutions for each
treatment and three samples at each dilution were
made. Flasks were incubated for 10 days, then fi xed
in methanol and stained with Giemsa. Colonies with
more than 50 cells were considered survivors.
Chromosomal Aberrations
Dicentrics and centric rings were recorded in fi rst
mitosis. After irradiation, cells were harvested from
the central part of dishes. At fi rst mitosis, a 2-hour
INACTIVATION AND CHROMOSOMAL ABERRATIONS
JEPTO 2004, Volume 23, Number 1 87
treatment with colcemid was applied in order to
accumulate mitotic cells. Th e cells were trypsinized,
incubated with hypotonic 75 mM KCL at 37 °C
for 20 minutes, then fi xed with a 3:1 mixture of
methanol:acetic acid. Th e slides were stained with
3% Giemsa, and the chromosomes were studied
under light microscopy.
Results
Cell Inactivation
In a frame of a model assigning the lethal eff ective-
ness of ultrasoft X-rays to the core ionization events
on DNA, a sharp increase in RBE for inactivation
(RBEinac) was predicted¹⁶¹⁷ for these particles from
below to above the C K-threshold (Fıg. 5). Two
campaigns of experiments indeed demonstrated
the existence of such an RBEinac enhancement.¹⁶¹⁸
Th e average value of RBEinac (350 eV)/RBEinac
(250 eV) is equal to 2.0 ± 20%.¹⁸ Also, RBEinac
(350 eV)/RBEinac(810 eV) is ~2.
Chromosomal Aberrations
Dicentric + ring chromosomal aberrations were
measured during two recent campaigns of experi-
ments at LURE. Th e relative effi ciencies (RBEaber)
of the 350 and 810 eV beams relative to the 250 eV
beam are, respectively:
RBEaber(350)/RBEaber(250) = 2.5 ± 0.5
RBEaber(810)/RBEaber(250) = 1.0 ± 0.2
Energy (eV)
200 400 600 800 1000
Ab
so
rpti
on
0.0
0.2
0.4
0.6
0.8
1.0
1.2
ONC
FIGURE 4. Ultrasoft X-ray absorption in a 1-µm slice of cell with spleen composition.
88 JEPTO 2004, Volume 23, Number 1
ARNAUD BOISSIÈRE ET AL.
Discussion and Conclusions
Th e RBEinac increase above versus below the C
K-threshold seems unambiguously related to that
(2.1) of core events on DNA per dose unit in cell.¹⁸
Th e ratio RBEinac (350 eV)/RBEinac(810 eV) ~2
may probably be interpreted in the same way. It
is noteworthy that the corresponding ratio of core
events on DNA per dose unit in the cell is 3.6.¹⁸
Th is might indicate a lethal effi ciency of core events
that is higher at 810 eV than at 350 eV. In view of
the higher energy of the involved electrons (Table
1), such an eff ect could be expected. Supporting this
hypothesis, the effi ciency of a core event on DNA to
induce DNA double-strand breaks in pBs plasmids
was found about two times larger at 760 eV than
at 380 eV.¹⁸
Ratios of RBEaber are found very close to those
for RBEinac. Th is suggests that core ionizations on
DNA might be initiating events for this second
end-point as well. Because of the large energy lo-
cally deposited by these events, they might induce
complex unrejoined DSBs or clusters of DSBs prone
to misrejoining, as already conjectured for ions.⁸ An
alternative hypothesis, based on a single DSB and a
recombinational repair mechanism,¹⁹ was proposed
to explain the overall large RBEaber of ultrasoft X-
rays. Th e connection between CA and core events on
DNA presented here may suggest new approaches
to the investigation of the involved mechanisms.
PK
PL
OK
Energy (eV)
100 1000 10000
RB
E
0
5
10
15
PK
PL
CK
NK
OK
FIGURE 5. Predicted enhancement of the RBE of ultrasoft X-rays from below to above the C K-threshold when the
lethal effectiveness is attributed to core events in DNA (1.2% lethal effi ciency assumed for P L- and C K-ionizations
[see Table 1]).
INACTIVATION AND CHROMOSOMAL ABERRATIONS
JEPTO 2004, Volume 23, Number 1 89
Acknowledgments
We are very grateful to A. Chatterjee for very stimu-
lating discussions. Th is work was supported by CEA
(LRC No. 6), CNES (contract No. 793/99), and
CNRS (contract PCV00-033). Th e experiments
were performed at the LURE facilities. Th e work
in L. Sabatier’s laboratory was supported by FIGH-
CT-1999-00003 (RADINSTAB).
References
1. Dikomey E, Dahm-Daphi J, Brammer I, Mar-tensen R, Kaina B. Correlation between cellular radiosensitivity and non-repaired double-strand breaks studied in nine mammalian cell lines. Int J Radiat Biol 1998; 73:269–278.
2. Natarajan AT, Obé G. Molecular mechanisms involved in the production of chromosomal aber-rations: I. Utilization of Neurospora endonuclease for the study of aberration production in G2 stage of the cell cycle. Mutat Res 1978; 52:137–149.
3. Bryant PE. Enzymatic restriction of mammalian cell DNA using PvuII and BamH l evidence for the double-strand break origin of chromosomal aberrations. Int J Radiat Biol 1984; 46:57–65.
4. Natarajan AT, Obé G. Molecular mechanisms involved in the production of chromosomal aber-rations. Chromosoma 1984; 90:120–127.
5. Lea DE. Actions of Radiations on Living Cells. 1 edition. Cambridge University Press, 1946.
6. Chen AM, Lucas JN, Simpsons PJ, Griffi n CS, Savage JRK, Brenner DJ, Hlatry LR, Sachs RK. Computer simulation of FISH data on chromo-some aberrations produced by X-rays or α particles. Radiat Res 1997; 148:S93–S101.
7. Radivoyevitch T, Hoel DG, Hahnfeldt PH, Rydberg B, Sachs RK. Recent data obtained by pulsed-fi eld gel electrophoresis suggest two types of double-strand breaks. Radiat Res 1998; 149:52–58.
8. Sachs RK, Chen AM, Simpsons PJ; Hlatry LR, Hahnfeldt P, Savage JKR. Clustering of radiation-produced breaks along chromosomes: modelling the eff ect of chromosome aberrations. Int J Radiat Biol 1999; 75:657–672.
9. Champion C. Structure des dépôts d’énergie des ions rapides dans l’eau. Application à l’étude par Monte Carlo de l’inactivation cellulaire. Th esis, Paris Pierre et Marie Curie University, 1999.
10. Champion C, L’Hoir A, Politis MF, Chetioui A, Fayard B, Touati A. Monte-Carlo simulation of ion track structure in water: ionization clusters and biological eff ectiveness. Nucl Instr Meth B 1998; 146:533–540.
11. Carlson TA, Krause MO. Relative abundance and recoil energies of fragment ions formed from the X-ray photoionization of N2, O2, CO, NO, CO2, and CF4. J Chem Phys 1972; 56:3206–3209.
12. Kobayashi K, Hieda K, Maezawa H, Furusawa Y, Suzuki M, Ito T. Eff ects of K-shell X-ray absorp-tion of intracellular phosphorus on yeast cells. Int J Radiat Biol 1991; 59:643–650.
13. Saigusa S, Ejima Y, Kobayashi K, Sasaki MS. In-duction of chromosome aberrations by monochro-matic X-rays with resonance energy of phosphorus K-shell absorption edge. Int J Radiat Biol 1992; 61:785–790.
14. Chetioui A, Despiney I, Guiraud L, Adoui L, Sa-batier L, Dutrillaux B. Possible role of inner-shell ionization phenomena in cell inactivation by heavy ions. Int J Radiat Biol 1994; 65:511–522.
15. Kraft G. Radiobiology of very heavy ions: inacti-vation, induction of chromosome aberrations and strand breaks. Nucl Sci Appl 1987; 3:1–28.
16. Hervé du Penhoat MA, Fayard B, Abel F, Touati A, Gobert F, Despiney-Bailly I, Ricoul M, Sabatier L, Stevens DL, Hill MA, Goodhead DT, Chetioui A. Lethal eff ect of carbon K-shell photoionizations in Chinese hamster V79 cell nuclei: experimental methods and theoretical analysis. Radiat Res 1999; 151:649–658.
17. Fayard B. Cassures d’ADN induites par ionisa-tion en couche interne et leurs conséquences bi-ologiques. Paris Pierre et Marie Curie University, thesis, 1999.
18. Fayard B, Touati A, Abel F, Hervé du Penhoat MA, Despiney-Bailly I, Gobert F, Ricoul M, L’Hoir A, Politis MF, Hill MA, Stevens DL, Sabatier L, Sage E, Goodhead DT, Chetioui A. Cell inactivation and double-strand breaks: the role of core ionizations, as probed by ultrasoft X rays. Radiat Res 2002; 157:128–140.
19. Savage JRK. A brief survey of aberration origin theories. Mutat Res 1998; 404:139–147.
