2. 226
at their Harwell Laboratories. It is produced by
iron-sulphur filtration of fission reactor neutrons
and has a high purity such that 79% of the neu-
tron kerma of the beam in water is due to 24-keV
neutrons (Perks et al., 1988). The neutron kerma
rate was 0.33 Gy/h with a y-ray contamination of
0.07 Gy/h (Perks and Goodenough, personal
communication).
Fresh whole blood from a healthy female donor
was collected at the start of the working day and
divided into 4-ml aliquots in four 25-cm3 T-steri-
lin flasks. These were irradiated sequentially dur-
ing the day by holding them vertically in a light
aluminium clamp in the 37 °C irradiation cham-
ber which has been previously described (Perks et
al., 1988). Throughout the time prior to irradiation
the samples were held in a 37 °C incubator. Fol-
lowing irradiation whole blood was cultured in
RPMI supplemented with fetal calf serum, L-
glutamine, antibiotics and PHA. 5-Bromo-2-de-
oxyuridine (10 /~g/rnl final concentration) was
added and cultures were grown at 37°C and
sampled at 68 h (1 h colcemid). After a mild
hypotonic treatment during harvest (Aghamoham-
madiet al., 1984), air-dried slides were harlequin
stained (Perry and Wolff, 1974). Coded and
randomised slides from duplicate cultures at each
treatment were scored for 3 end-points:
(a) All categories of chromosome-type aberra-
tions were scored in a random sample of complete
(46 centromeres) first-division cells.
TABLE 1
(b) MN were scored in a sample of blast cell
interphases (cells with large nuclei and delimited
cytoplasm). No attempt was made to distinguish
cells that had divided from those that had not,
since differential staining from BrdU uptake was
not sufficiently pronounced in the interphase
nuclei (Pincu et al., 1984).
(c) SCE and centromeric colour switches (CS)
were scored in second-division metaphases.
Results
The results are summarised in Tables 1 and 2
and shown graphically together with statistically
fitted curves in Figs. 1 and 2. All doses have been
expressed as kerma in water. Absorbed doses in
whole blood will be somewhat smaller, by a factor
primarily proportional to the relative hydrogen
contents. Lloyd et al. (1988) have estimated this
factor to be 0.89.
As expected all categories of chromosome-type
aberrations resulted from irradiation. For presen-
tation they have been grouped into dicentrics,
dicentrics plus centric rings and acentric frag-
ments (interstitial plus terminal deletions and ex-
cluding all exchange-related fragments). All 3 cat-
egories increased linearly with dose, and the fitted
regressions are shown in Fig. 1. However, a slight
positive curvature cannot be ruled out as both
quadratic and power law models provided accep-
table fits to the data. For the quadratic fits, the
PRODUCTION OF CHROMOSOME-TYPE ABERRATIONS, MICRONUCLEI AND SISTER-CHROMATID EXCHANGES
BY 24-keV NEUTRONS IN HUMAN G o LYMPHOCYTES
Neutron Chromosome aberrations Micronuclei Sister-chromatid exchanges
kerma Number AF/cell (D + R)/cell D/cell Total Number Per cell Number CS/cell Range Mean
in water of Ab/ of of SCE/ SCE/
(Gy) cells cell cells cells cell cell
0 70 0 0 0 0 3000 0.008 140 1.529 1-12 5.786
( + SE) (1.67 x 10 - 3) (0.10) (0.20)
0.33 57 0.35 0.33 0.30 0.68 4000 0.0915 73 1.836 1-12 6.726
( + SE) (0.07) (0.07) (0.07) (0.11) (4.78 × 10- 3) (0.16) (0.30)
0.66 49 0.65 0.98 0.75 1.63 4000 0.141 76 1.513 4-18 7.855
( + SE) (0.11) (0.14) (0.12) (0.18) (5.90 × 10 - 3) (0.16) (0.32)
1.00 58 1.10 1.36 1.14 2.46 4000 0.233 102 1.461 3-15 7.333
( + SE) (0.14) (0.15) (0.15) (0.20) (7.64 x 10 - 3) (0.12) (0.27)
AF, acentric fragments; D, dicentrics; D + R, dicentrics + centric rings.
4. 228
TABLE 2
PARAMETERS OF EQUATIONS FITTED TO DOSE-RESPONSE CURVES WITH 24-keV NEUTRON IRRADIATION IN G o
HUMAN LYMPHOCYTES
Linear ( y = c + aD ) Quadratic (y = c + aD + bD 2) Power law y = c + kD"
c a (Gy -1) P for c a (Gy -1) b (Gy 2) P for c k (Gy-') n P for
fit fit fit
Aberrations
AF 0 1.062 * * * 0.88 0 0.977 0.110 0.68
(0.099) (0.310) (0.378)
D 0 1.099 * * * 0.67 0 0.872 0.290 0.58
(0.100) (0.317) (0.392)
D + R 0 1.338 * * * 0.36 0 1.036 0.385 0.23
(0.111) (0.355) (0.441)
MN 0.009 0.220 * * * 0.19 - - -
(0.003) (O.OLO)
SCE 5.951 1.810 * * 0.14 5.745 4.899 - 3.242 * * 0.35
(0.282) (0.498) (0.277) (1.718) (1.734)
CS 1.611 -0.092 ** 0.15 1.553 0.646 -0.749 ** 0.16
(0.097) (0.159) (0.105) (0.606) (0.594)
0 1.080 1.052 0.82
(0.126) (0.232)
0 1.162 1.184 0.43
(0.131) 0.235)
0 1.425 1.212 0.32
(0.146) (0.214)
Significance: * P < 0.05; **P < 0.01; ***P < 0.001.
P = goodness of fit to linear or quadratic line.
curvature could not be ruled out) and this is in
accord with both expectation for high-LET par-
ticles in general, and with previous work with this
radiation quality (Lloyd et al., 1988; Roberts et
al., 1987). There are slight discrepancies in the
absolute values between Lloyd's data and ours, a
factor of - 33% for all categories (when doses are
expressed either in terms of kerma in water or
absorbed dose in blood). The reasons for this
discrepancy remain unclear but will be investi-
gated further by the two laboratories.
Having obtained a positive response for asym-
metrical chromosome-type aberrations, it is hardly
surprising to find one also for micronuclei, the
bulk of which arise from acentric fragments ex-
cluded from daughter cells at anaphase. No weight
can be put on absolute values here, because we are
not able, with the experimental protocol em-
ployed, to isolate for scoring, a cohort of cells
which had divided once and once only. Conse-
quently some of the cells scored will be undivided
and therefore cannot have MN (other than spon-
taneous ones) and others (a selected group) will
have divided more than once and have either
augmented or diminished MN frequencies. The
observed reduction factor, R - 0.1 must therefore
be interpreted with caution, but it does emphasise
the inefficiency of MN, relative to aberrations for
detecting cytogenetic damage. The value is also in
line with a low probability of fragment exclusion
(PE- 0.2) generally characteristic of lymphocytes
(Savage, 1988).
Although the SCE increase is not dramatic, it is
significant and there is evidence of a positive dose
response. This agrees with some of the more re-
cent findings that high-LET, in contrast to low-
LET radiations, are able to induce SCE in un-
stimulated lymphocytes (Savage and Holloway,
1987; Aghamohammadi et al., 1988).
Unlike the other two end-points, SCE produc-
tion does not appear to be linear over the whole
dose range (although an acceptable straight line
can be fitted) but there is a tendency to saturate at
higher doses. This is similar to our finding for
plutonium a particles, but not for 42-MeV neu-
trons. The reasons for saturation are not, at pres-
ent, obvious. Whilst interphase 'death' (the failure
of irradiated cells to reach mitosis) is a very real
5. phenomenon in unstimulated lymphocyte popula-
tions both in vitro and in vivo (Lloyd et al., 1973;
Savage and Breckon, 1985), there does not appear
to be a massive selection against cells with high
conventional aberration yields, and one might an-
ticipate that such would also be the case for SCE,
which are usually considered selectively neutral.
Recent observations of under-dispersed SCE dis-
tributions (Aghamohammadi et al., 1988; Moquet
et al., 1987) might suggest otherwise, but no such
distributions were found in the present studies.
The very short recoil protons from 24-keV neu-
trons would not be expected to produce the sub-
stantial correlation of damage along single tracks
which occurs with the passage through cells of
other high-LET radiations, such as slow a par-
tides (Goodhead et al., 1980; Edwards et al.,
1980).
The differential delay and perturbation intro-
duced by the different dose increments means
that, by using a single sampling time, we may be
analysing quite different second-division cell
populations. If there is any element of heterogene-
ity or change of frequency with time in target
cells, then this will be reflected in the average
yields in the different cell mixtures scored, and
distorted dose-response curves are to be expected.
We do not have data from other sample times to
test this, but note that any heterogeneity present
was insufficient to produce over-dispersion in the
between-cell SCE distributions. The ability to de-
fine a narrow cohort of target cells which could be
recognised and selected for scoring irrespective of
delay, would offer considerable advantages in a
situation like this.
If we neglect the highest dose, then the fitted
linear regression for SCE becomes Y= 5.771 +
3.102 (_+0.755)D i.e.- 3 induced SCE/cell/Gy.
This rate of induction at low doses (< 0.66 Gy)
can be compared with -10 SCE/cell/Gy for
plutonium a particles (Aghamohammadi et al.,
1988) and 3.2 SCE/cell/Gy for 42-MeV neutrons
(Savage and Holloway, 1988). X-Rays were not
tested in this experiment, but all our previous
findings indicate that they are unable to produce
SCE when delivered to unstimulated lymphocytes.
So, for this quality of neutrons also, the RBE for
SCE remains undefined and effectively infinite.
229
Acknowledgements
We wish to thank UKAEA for providing radia-
tion facilities of 24-keV neutrons and the reactor
staff at UKAEA for their co-operation in irradiat-
ing the blood samples. We are also grateful to
David Papworth for his statistical advice and dis-
cussions.
This work was supported by the Commission of
the European Communities Contracts No. B16-E-
164-UK and No. B16-A-009-UK.
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