Download Lethality of radioisotopes in early mouse embryos

/. Embryol. exp. Morph. Vol. 52, pp. 203-208, 1979
203
Printed in Great Britain (g) Company of Biologists Limited 1979
Lethality of radioisotopes in early mouse embryos
By HILARY A. MACQUEEN 1
From ihe Imperial Cancer Research Fund,
London
SUMMARY
The development of pre-implantation mouse embryos was found to be prevented by
exposure of the embryos to [35S]methionine, but not to [3H]methionine. Such embryos
have also been shown to be highly sensitive to [3H]thymidine. These observations
are discussed with reference to the path lengths and energies of electrons emitted
from the different radioisotopes.
INTRODUCTION
In order to follow the expression of stage-specific proteins during early
mammalian development, a number of investigators have incubated preimplantation mouse embryos in vitro in medium containing [35S]methionine
(Van Blerkom, Barton & Johnson, 1976; Dewey, Filler & Mintz, 1978;
Levinson, Goodfellow, Vadeboncoeur & McDevitt, 1978). Concentrations of
[35S]methionine used have varied between 50 and 200 /tCi/ml, and incubation
times have been of the order of 1-2 h. In the course of similar experiments
where the fate of embryos was followed after labelling, I have found that
embryos which had been exposed to 50 /*Ci/ml [35S]methionine for as little as
30 min would always degenerate and die within a few hours. A study was
therefore undertaken of the factors affecting the response of early mouse
embryos to radioisotopes, and the work has been extended to the response of
undifferentiated teratocarcinoma cells which appear to have a similar sensitivity
to radiation.
MATERIALS AND METHODS
Embryos at the 2- to 4-cell stage (1-5 days post coitum) were flushed from
the oviducts of C3H/He mice and cultured in M16 medium (Whittingham,
1971) at 37 °C in an atmosphere of 5 % CO2 in air.
The cells lines used were A6, a pluripotent murine teratocarcinoma cell
line originally derived from the OTT 6050 ascites tumour, and STO, a murine
fibroblast line (Hogan, 1976). A6 was grown in Dulbecco's modified Eagle's
1
Author's address: Imperial Cancer Research Fund, Mill Hill Laboratories, Burtonhole
Lane, London, NW7 IAD. U.K.
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H. A. MACQUEEN
medium (DMEM) supplemented with 10 % calf serum and 10 % foetal bovine
serum; STO was grown in DMEM supplemented with 10% calf serum. Both
were maintained in an atmosphere of 5 % CO2 in air at 37 °C. The modal
chromosome number of A6 is 40, that of STO 64.
L-[35S]methionine (1100 Ci/mmol, 5-95 mCi/ml), L-[methyl-3H]methionine
(12 Ci/mmol, 1 mCi/ml) and [methyl-^CJthymidine [56 mCi/mmol, 50/*Ci/ml)
were from the Radiochemical Centre, Amersham, England. Radioactive
methionine was freeze-dried prior to use and resuspended in an appropriate
volume of medium. Embryos were labelled in M16 medium, and cultured cell
lines were labelled in a medium consisting of 80 % methionine-free DMEM,
10 % complete DMEM and 10 % dialysed calf serum. This gave good uptake
of radioactive methionine. Radioactive thymidine was added directly to the
culture medium.
To measure incorporated radioactivity, embryos were transferred onto
Whatman GF/C filter discs (2-4 cm diameter) and extracted with ice-cold
5 % trichloracetic acid for 10 min with shaking. The filters were then washed
in ethanol, dried at room temperature, and counted in a Packard Tri-Carb
liquid scintillation spectrometer, using 0-6 % diphenyl oxazole (PPO) and
0-075 % l,4-bis-(2-(4-methyl-5-phenyloxazolyl))-benzene (POPOP) in toluene.
A6 cells were exposed to 0-1 /^g/ml colcemid for 2 h, and their chromosomes
prepared by a standard air-dry procedure after fixation with 1:3 methanol: acetic
acid. Chromosomes were stained with Giemsa and photographed with a Zeiss
Photomicroscope Mark III.
RESULTS AND DISCUSSION
Experiments to examine the synthesis of stage-specific embryonic proteins
revealed that exposure to [35S]methionine prevented further division of the
embryos. To investigate this effect of [35S]methionine on development, 2 to
4-cell embryos were incubated for 2 days in M16 containing different concentrations of the radioactive amino acid. At the end of this time normal,
control embryos had undergone two rounds of cell division and compaction
had begun. Embryos were therefore scored as abnormal if they consisted of
fewer than eight cells; in most cases these abnormal embryos contained one
or more degenerating blastomeres. As shown in Fig. 1, normal development
is affected at about 1 /*Ci/ml [35S]methionine, and at concentrations above
7 /tCi/ml none of the embryos become compacted morulae. Although a detailed
study of relationships between the effect of [35S]methionine and stage of the
cell cycle was not made, it was noted that a few embryos in each experiment
were able to undergo one division. This suggests that there is a point in the
cell cycle beyond which exposure to [35S]methionine will not prevent mitosis.
If the embryos are being killed by decay of the incorporated [35S]methionine,
rather than by contaminants in the radioactive methionine, for example, it
should be possible to relieve the effect by reducing the specific activity of the
205
Embryo killing by radioisotopes
0-1
35
1-0
10
S-Mctliioninc uCi,
100
Fig. 1. Effect of varying concentrations of [35S]methionine on embryo survival.
Survival of the control embryos, which had not been exposed to [35S]methionine,
was 90-100 % in different experiments. Each experimental group contained
between 10 and 30 embryos. Number of replicate experiments: 5.
label. This was achieved by the addition of non-radioactive methionine to
the culture medium. Results of such an experiment are shown in Table 1. It is
clear that survival can indeed be increased by lowering the specific activity to
002 Ci/mmol. Furthermore, it could be shown, in separate experiments, that
exposure for as little as 30 min to 50 JLLQ/ml (1100 Ci/mmol) is sufficient to
cause subsequent death of the embryo. Interestingly, the killing effect is specific
for [35S]methionine: labelling with [3H]methionine at the same specific activity
(12 Ci/mmol) has no effect on embryo viability even at concentrations as high
as 200 /tCi/ml (Table 1).
We have also studied the effect of [35S]methionine on undifferentiated murine
teratocarcinoma stem cells, which are analogous in many ways to embryonic
cells, in particular to cells of the embryonic ectoderm of the 5-day post coitum
mouse embryo (Martin, 1978). As shown in Fig. 2, concentrations of 10 and
100 /tCi/ml [35S]methionine (1100 Ci/mmol) caused cell lysis of teratocarcinoma
cells over a period of 2 days, but had much less effect on STO murine fibroblasts. When the chromosomes of teratocarcinoma cells exposed to 5 /tCi/ml
[35S]methionine for 24 h were examined, numerous chromatid breaks and
chromosome rearrangements were seen (Fig. 3). Such breaks were not seen
in control cells that had not been exposed to radioactive methionine.
Snow has shown that labelling embryos with [methyl-3H]thymidine at concentrations as low as 0-05/tCi/ml (27 /tCi/mmol) will result in cell death
(Snow, 1973). The relative lethality of these different isotopes to embryonic
cells is approximately what would be expected from the differing properties
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EMU 52
206
H. A. MACQUEEN
Table 1. The effect of various treatments on embryo development. Survival and
counts per minute per embryo were measured after 2 days in culture. Data
pooled from 15 experiments
Final
concentration
of added
methionine
Treatment
Og/ml)
None
10/tCi/ml [35S]methionine
12/tCi/ml [35S]methionine
10/tCi/ml [35S]methionine
12 /tCi/ml [3H]methionine
200/tCi/ml [3H]methionine
Specific
Number
of
activity
(Ci/mmol) embryos
0
00014
1100
015
12
70
0-02
015
12
12
2-5
43
15
11
15
23
25
A
/o
Survival
98
0
0
27
97
100
Counts per
minute per
embryo
0
3876
651
33
55
412
B
6
10 -
5 10s -
m
1
I
I
I
2 0
1
Time of exposure (days)
Fig. 2. Effect of [35S]methionine on the multiplication of cells on dishes. Cells
were removed from the dishes with trypsin, and counted using a haemocytometer.
Each point is the mean of three dishes. (A) A6 teratocarcinoma cells; (B) STO
fibroblasts. • — • , Control, no radioactive label; # — # , 10/^Ci/ml [35S]methionine; O—O, 100/MCi/ml [35S]methionine. Number of replicate experiments = 4.
of their electrons. The probability that an electron produced by radioactive
decay interacts with nuclear DNA will depend on the site of the disintegrating
atom and the energy and path length of the emitted electron. Weak /3~ emitters
such as 3 H should be lethal only if located in the nucleus. Stronger fi~ emitters,
such as 35S or 14C, producing electrons with higher energy and longer path
length which can reach the nucleus from any part of the cell, can therefore be
lethal wherever they are located; in fact because the density of ionizations
produced by their electrons is low near the start of their paths the decay in
the cell nucleus of an isotope such as 35S or 14C will produce fewer ionizations
in that nucleus than the decay of tritium (Tagder & Scheuermann, 1970) and
Embryo killing by radioisotopes
207
<fc
(J
Fig. 3. Metaphase spread of A6 cells exposed to 5/*Ci/ml [35S]-methionine
Chromatid breaks are marked with small arrows, an inter-chromosomal rearrangement with a large arrow. Chromosome number = 80. About one cell in 20 had
80 chromosomes; the modal chromosome number = 40.
therefore produce less damage (Garder & Devik, 1963). Indeed, in unpublished
experiments I have found that [14C]thymidine is not lethal to embryos, even
at concentrations and specific activities higher than those used by Snow
(1973) in his experiments with [3H]thymidine.
It is not surprising that mouse embryos are sensitive to irradiation from
/?- emitters in view of their sensitivity to X-rays (Russell, 1957; Rugh & Grupp,
1961; Ohzu, 1965; Goldstein, Spindle & Pedersen, 1975). The basis for this
sensitivity, and the similar sensitivity of teratocarcinoma cells, is not known.
One possibility is that the cells are deficient in recombinational repair, because
teratocarcinoma cells show a reduced frequency of sister-chromatid exchanges
(F. Kelly, personal communication). However, it should be pointed out that
STO cells may be more resistant to [35S]methionine because they have more
chromosomes, rather than because they have more efficient repair mechanisms
than A6 cells.
In view of the death of early mouse embryos and teratocarcinoma cells
after exposure to radiation, it is suggested that some caution be exercised in
the interpretation of data from experiments involving radioisotopes, particularly
[35S]methionine.
I should like to thank Drs Brigid Hogan and John Cairns for much helpful discussion
and critical reading of the manuscript, and Jane Tate for skilled technical assistance.
208
H. A. MACQUEEN
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DEWEY,
(Received 24 January 1979, revised 13 February 1979)