Download Genetic susceptibility to radiation effects in early embryos

GENETIC SUSCEPTIBILITY TO RADIATION EFFECTS IN EARLY
EMBRYOS
P. Jacquet, Laboratory of Radiobiology, Belgian Nuclear Research Centre,
B-2400 Mol
1. Introduction
It has been known for a long time that the tissues in intense proliferation, i.e. those
containing many dividing cells, are particularly radiosensitive. This is the law
formulated as early as in 1906 by Bergonié and Tribondeau, after which the
radiosensitivity of a tissue increases as its cells are less differentiated, have a greater
potential of proliferation and divide more rapidly. Therefore, it can be supposed that
the embryo should constitute a particularly vulnerable target for radiation, specially
during the first steps of development.
Among the somatic effects of radiation other than cancer, developmental effects on
the unborn child are of greatest concern. The principal factors of importance for the
induction of developmental effects are the dose and the stage of gestation at which it
is delivered. Dose rate is also of significance, since many pathological effects on the
embryo are reduced significantly by reducing the dose rate.
Most experimental data on the effects of radiation in the developing embryo or fetus
have been obtained with the mouse or the rat, animals that reproduce in quantity with
a relatively short gestation period. The principal events in the development of an
embryo,
namely,
cleavage,
implantation,
placentation,
organogenesis,
and
differentiation of the various organs, tend to occur in all mammals in roughly the
same sequence; it is the time scale that differs. It is probably justified, therefore, to
assume that the major effects seen in the mouse or rat when irradiation is delivered at
specific stages of development will also occur in humans at the equivalent stages.
Embryonic development is usually divided into three periods, each of them showing a
characteristic sensitivity to ionizing radiation and other external agents.
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The preimplantation period extends from the fertilization up to the implantation of
the embryo into the uterine walls. Its duration (almost one week) does not greatly
differ between the various mammalian species.
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Implantation consists into the attachment of the embryo to the uterus, its
penetration through the epithelium and the beginning of the complex interactions
of the embryo with its mother. Implantation is followed by the organogenesis
period, during which organs are formed according to a well defined sequence for
each species. In humans, this period extends from the 2nd to the 8th week of
pregnancy. Near its end, the embryo measures about 30 millimeters and weighs
2-2.7 grammes.
-
The foetal period represents nearly 70 % of the total pregnancy in humans. It is
the period of general growth and functional maturation of the newly formed
organs. Important developments occur during this period, for example
neurogenesis and synaptogenesis, and formation of the external genital organs.
This paper will first summarize the “classic” effects of an exposure of the mammalian
embryo to ionizing radiation during the three above mentioned periods. It will then
concentrate on some unexpected results obtained during recent years, following
irradiation of mouse embryos during the very early stages of development. In humans,
these early stages are practically untraceable as they pass before the most sensitive
radioimmunoassay tests could detect an increased concentration of human chorionic
gonadotrophin (hCG) in the urine, indicative of trophoblastic activity after
implantation. Therefore, an estimation of the potential risks of an exposure between
fertilization and early postimplantation must exclusively rely on data from animal
experiments. The recent results obtained in this field could be of potential concern for
radiation protection.
2. Classic effects of ionizing radiation on the developing organism
2.1. The preimplantation period
Lethality has been recognized as the main effect of irradiation during the
preimplantation period (Figure 1). It has also long been assumed that, following
irradiation, early embryos would either die or survive without any detectable
malformations : this was the well-known “all-or-none-rule” formulated as early as in
1956 by Russell and Russell.
Studies performed with the aid of in vitro techniques, and in which we were involved,
showed that:
1) Sensitivity to the killing effects of radiation is higher during early stages. It could
be estimated that, at the time of highest sensitivity, i.e. the one-cell stage a few hours
after fertilization, the mortality would increase of about 1 % per 10 mGy of acute Xirradiation.
2) For the same embryonic stage, sensitivity can, however, vary by a factor 10,
depending on the position of the cells in the cell cycle.
3) Embryonic mortality following irradiation during the preimplantation stages occurs
predominantly near the time of implantation, and results mainly from structural and
numerical chromosome aberrations.
Figure 1 : Incidence of abnormalities and of prenatal and neonatal death in mice given
a dose of 2 Gy at various times after fertilization (redrawn by Brent, from L.B.
Russell and W.L. Russell : J. Cell Physiol. [Suppl. 1] 43, 103, 1954). The lower scale
consists of Rugh's estimates of the equivalent stages for the human embryo.
2.2. The organogenesis period
During the organogenesis period, the main effect of radiation in small rodents is the
production of a variety of congenital abnormalities (Figure 1).
For each species, there exists a well determined period of sensitivity to the induction
of each malformation. Increasing the dose usually results in an extension of this
period of sensitivity and in an increase of the incidence of malformations. The
specific time when a malformation is produced coincides with the main stage of
differentiation and organization of the considered structure. The experimental data
show that the form of the dose-effect relationship for the induction of malformations
is generally sigmoid, the frequency of malformations by unit of dose increasing with
the dose. Experiments performed in rodents suggest that 100 mGy represents the
minimal dose to induce malformations in organs.
Embryos exposed during early organogenesis also show the greatest intrauterine
growth retardation, expressed as a weight reduction at term. This phenomenon results
from cell depletion. Animals show a remarkable ability to recover from growth
retardation induced by irradiation during organogenesis, and while they may be
smaller than usual at birth, they may achieve a normal weight as adults.
Contrary to what is observed in experimental animals, radiation-induced
malformations have been rarely seen in humans. This difference could be the result of
two factors. First, in rats and mice carefully conducted experiments have been
performed, with exact control of dose and precise timing. In humans, by contrast, the
limited data that are available have resulted from the random irradiation of relatively
few individuals. Second, the period of organogenesis, when a range of gross
anomalies may be produced, represents only 15 % of the total duration of pregnancy
in humans. On the other hand, the development of the central nervous system is taking
place for much of the long foetal period in human pregnancy, meaning that this
system is the most likely target for radiation-induced damage.
2.3. The foetal period
The consequences of an exposure of mammalian embryos to ionizing radiation during
the foetal period are usually much less spectacular. It can induce anomalies in the
development of the tissues (since histogenesis is much active at that time) and
generalized or localized growth retardation. In contrast to what is observed after
irradiation during organogenesis, the growth retardation induced during the foetal
period frequently persists during all the extra-uterine life. Various other effects have
been described in laboratory animals, including effects on the hematopoietic system,
liver and kidney, all occurring, however, after fairly high radiation doses. The effects
on the developing gonads have been particularly well documented, both
morphologically and functionally. Doses of a few hundreds milligrays are necessary
to produce fertility changes in various animal species.
However, the exposure of the human conceptus during the foetal period may lead to a
diminution of the IQ, associated or not with microcephaly. Such effects have been
predominantly observed in survivors of the atomic bombings of Hiroshima and
Nagasaki who had been exposed in utero between the 8th and 15th weeks of
pregnancy. The data suggest the possibility of a non-threshold type response,
interpreted as representing a loss of 3 IQ points per 100 mGy of X-rays or gamma
rays (30 IQ points per Gy).
3. Sensitivity of the preimplantation embryo to external agents : the dogma of
Teratology revisited?
3.1. Recent results following irradiation
As mentioned in 2.1., it has long been admitted that embryos escaping killing by an
irradiation (or any other agent) during the preimplantation period would develop
without anomaly. The logic of this “dogma” of Teratology was that the embryonic
cells are still undifferentiated at these stages and that loss of one or a few cells could
be compensated by other cells. It must be underlined, however, that already in the ’60,
the team of Rugh had reported about exencephalies and dwarfism after exposure of
mouse CF1 preimplantation embryos to low doses of X-irradiation. These results had
provoked a lot of criticism, essentially because of the lack of a clear dose-response
relationship and of sound control data. Less known is the fact that Rugh and his
colleagues also reported that 1 Gy of X-rays to the newly fertilized egg of the CF1
strain had caused as much as 98% of the males and 97% of the females surviving to
18 months of age to develop cataracts. Only 13% and 17% of the control males and
females, respectively, had developed this defect at the same age.
Near the end of the eighties, definite malformations were obtained in mouse foetuses
of the Heiligenberger strain that had been irradiated during the preimplantation
period, obliging the scientific community to reconsider the “dogma” of Teratology.
The results obtained in Germany showed that there was a strong mouse strainspecificity in the sensitivity to the teratogenic effects of radiation during the
preimplantation period and pointed to the peculiar sensitivity of the one-cell embryo,
or zygote. The main malformation induced by irradiation of preimplantation embryos
of the Heiligenberger strain, 1 hour after fertilization, was gastroschisis. This
malformation was also encountered in unirradiated embryos, but its frequency was
very clearly increased by irradiation with X-rays or neutrons. This suggested that the
frequency of malformations could be increased by irradiation in those mouse strains
already showing a specific predisposition for these malformations. There were
marked differences in the sensitivity of the various preimplantation stages, the onecell stage being the most sensitive, but there was no time during the preimplantation
development that did not show an increased teratogenic risk. The data were also
compatible with an absence of threshold dose for the induction of malformations
during the one-cell stage.
Since that time, Gu and colleagues, in Japan, also reported about malformations in
mouse ICR embryos that had been gamma-irradiated at various preimplantation
stages. Like in Germany, the highest effects were obtained after irradiation of one-cell
embryos, in this case 2 hours after fertilization, but other preimplantation stages were
also sensitive. In contrast to those, however, the malformations observed were very
variable and included exencephaly, cleft palate, chest hernia, abdominal hernia, open
eye, anophtalmia, abnormal tail and polydactyly. No external malformations other
than open eye were observed in unirradiated fetuses. The average incidence of all
types of external malformations among mice irradiated at 2 h postconception with 0,
0.1, 0.25, 0.5 and 1.0 Gy were 0.19, 0.49, 3.1, 6.1 and 4.2 %, respectively. The
increase was significant from the dose of 0.25 Gy. The decrease observed at 1.0 Gy
was attributed to the fact that, at that dose, the lethal effects were dominant, resulting
into a probable elimination of a number of abnormal embryos. Another interesting
point of that study was that the susceptibility to external malformations appeared even
higher in pre-implantation stages than during organogenesis : a dose of 0.5 Gy given
on day 8 of gestation induced only exencephaly and exophtalmia, and the incidence of
external malformations induced by that dose did not differ from that observed in
unirradiated mice.
In our laboratory, we X-irradiated mouse zygotes 7 hours after fertilization and
obtained some evidence of teratogenic effects in the CF1 strain (used by Rugh in the
’60), but not in the BALB/c strain. The abnormalities observed in the CF1 strain
included exencephaly, polydactyly, hypodactyly and gastroschisis. In unirradiated
animals, only 1 of 862 fetuses showed exencephaly, and there were no other
malformations. Although the incidence of malformed fetuses was always low, it
showed a tendency to increase with the dose of radiation, from 0.12 % in the
unirradiated group to 2.27 % in the group irradiated with 1.0 Gy, the difference with
controls being significant for the doses of 0.1, 0.5 and 1.0 Gy. In addition to external
malformations, we also observed a number of underdeveloped or "dwarf" fetuses. The
incidence of dwarfs increased significantly following irradiation with 0.5 and 1.0 Gy.
When dwarfs were added to the malformed fetuses to calculate the incidence of
abnormal fetuses, the proportion of those increased from 1.8 % in the unirradiated
group to 5.2 % and 5.7 % in the groups irradiated with 0.5 and 1.0 Gy, respectively.
Like in the Japanese study, the number of surviving fetuses was very low in females
given 1 Gy, and it is possible that a number of abnormal embryos of this group had
been eliminated soon after implantation.
The group of Rutledge in the United States performed similar experiments in hybrid
mice (SEC X C57BL), that were given 1.5 or 2 Gy of X-rays 2.5 h after fertilization.
The anomalies found in the fetuses were mainly hydrops or generalized fetal edema,
wall defects such as gastroschisis, eye defects, exencephaly and cleft palate. Except
for the latter, these anomalies were also found in the control fetuses, but their
proportions increased from 1.2 %
to 3.6 % and 6.2 %, repectively, in groups
irradiated with 1.5 Gy and 2.0 Gy. The increase was marginally significant for the
dose of 2.0 Gy.
Another effect of irradiation during the preimplantation period is worthwhile. Indeed,
beside malformations, X-irradiation of zygotes revealed also able to induce a genomic
instability in surviving embryos. Thus, studies performed in Germany showed a dosedependent increase of chromosomal aberrations in Heiligenberger foetuses that had
been irradiated 1 h after fertilization. The chromosomal aberrations had developed
many cell generations after irradiation. They were even observed in morphologically
normal foetuses but were more frequent in the malformed ones. Furthermore, the
genomic instability was also observed in foetuses from another mouse strain (the
C57BL strain) carrying no predisposition to congenital malformations, that had been
similarly irradiated at the zygote stage. This suggests that the genomic instability
would be a phenomenon of a more general nature. Its consequences remain to be fully
determined. The radiation-induced genomic instability was also shown to be
transmitted to the following generation.
3.2. Recent results following exposure to chemicals
In view of the controversy caused by the few positive results obtained after irradiation
of pre-implantation embryos, an examination of some recent results obtained by
teratologists using chemical compounds will be instructive.
The laboratories of Generoso at Oak Ridge and Rutledge in Seattle have been
involved in a decade of study of mutagen effects on zygotes of hybrid mice.
Treatment of the female mice 1 to 9 h post-mating with a variety of short-lived agents
revealed to result into various unusual effects. Thus, a number of mutagens induced
embryonic lethality at different stages of gestation, including mid and late gestation
and even stillbirths or death before weaning. Furthermore, many chemicals revealed
also able to induce malformations after zygotic treatment. The malformations
observed in 17-18 day fetuses after treatment of the zygotes form a special class that
differs from the set induced during organogenesis, in that the defects are more
restricted in nature : hydrops (generalized fetal edema), bent limbs and tail, abdominal
wall defects and eye defects represent the majority of anomalies induced by such
treatments. Interestingly, exencephaly and digital defects have not been observed after
treatment of the zygotes by chemicals, although those have been reported in various
papers following irradiation. The list of chemicals able to induce malformations after
exposure at the one-cell stage includes : ethylene oxide, ethyl methanesulfonate,
diethyl
sulfate,
dimethyl
sulfate,
acrylamide,
ethylnitrosourea
and
triethylenemelamine.
A set of chemicals have also been shown to produce changes in the post-zygotic preimplantation embryo, resulting in embryonic death and malformations such as limb
defects, cleft palate, exencephaly, open eyelids, skeletal defects and digital numerical
aberrations (poly- or hypodactyly). The list of such agents includes :
methylnitrosourea, cyclophosphamide, ethylnitrosourea, retinoic acid (vitamin A) and
5-azacytidine. Importantly, not all of the effects were manifest in utero. Eighty four to
90 h old blastocysts were exposed in vitro to methylnitrosourea, transplanted to
surrogate mothers and allowed to go to term. While no malformations, radiographic,
morphometric, karyotypic or histologic defects were detected, the pups from the
treatment group had excess perinatal mortality and 58 vs. 22% mortality at one year.
4. Recent results on the radiation sensitivity of early postimplantation stages
4.1. Influence of the p53 gene on the embryonic radiation sensitivity
Early postimplantation development in mammals is associated with a dramatic
increase in the proliferation rate of undifferentiated stem cells that form the primary
embryonic layers, ectoderm, endoderm and mesoderm, and with the start of
differentiation of the embryo. Gastrulation occurs during the second week of
pregnancy in humans, ie at the limit of detection of pregnancy.
Various experiments have shown that the genetic constitution of the embryo could
interfere with its radiation sensitivity. Apoptosis (also defined as “cell suicide” or
"programmed cell death") is an essential physiological process in the normal
development of embryos, either for eliminating abnormal embryonic cells or for
controlling the number of developing embryos in various species. The p53 gene (“the
genome guardian”) plays a key role in the apoptotic process. In humans, germline
mutations in this gene cause an inborn predisposition to cancer, known as the “LiFraumeni syndrome”. Mice carrying such mutations have a decreased longevity and
an increased tumor incidence. They also show developmental effects such as an
increased embryonic and postnatal death and a higher level of malformations, in
which exencephaly and anencephaly predominate. Interestingly, the malformations
mostly affect the female embryos.
Whether mutations in the p53 gene could also increase the radiation sensitivity of
early embryos is an important question. In our laboratory, we mated p53 heterozygous
mice and X-irradiated the embryos with 0.5 Gy 7 days after fertilization,
corresponding to the gastrula stage. In both irradiated and control groups,
developmental abnormalities were found, affecting mainly the female homozygous
null embryos and, at a lesser degree, the heterozygous ones*. The proportion of
abnormal embryos was, however, significantly increased in the irradiated group (23.4
% vs. 12.9 % in controls). In the control group, the abnormalities consisted in
exencephaly and dwarfism. In the irradiated group, gastroschisis, polydactyly, cleft
palate and cephalic edema were also found.
These results point to the importance of the p53 tumour-suppressor protein for normal
development. They also clearly show that homozygous p53-/- (or heterozygous p53+/at a lesser extent) embryos may be more at risk for radiation-induction of external
malformations during the early postimplantation stages, and that such embryos are
able to survive to birth.
____________
*
According to Mendel's law, mating of a p53+/- (= heterozygous) female with a p53+/- male will
theoretically give 50 % p53+/- embryos, 25 % p53+/+ (= homozygous) and 25 % p53-/- (= homozygous
null) embryos.
4.2. Influence of other genes on the embryonic radiation sensitivity
The number of genes required to control the normal functions of mammalian cells and
organisms has been estimated to more than 30,000. To monitor damage and to
maintain the genes without significant alteration is a major concern for the cell, and
repair processes have evolved in all organisms to correct errors made in replicating
the genes and to restore damaged DNA. The consequences of loss of repair capacity
can be seen in a number of human syndromes showing hypersensitivity to
environmental agents. These syndromes generally show multiple symptoms, including
cancer-proneness, neurological disorders and immune dysfunctions. An important
question is whether individuals affected from such mutations are also more
susceptible to spontaneous or radiation-induced developmental effects.
Investigations performed todate with mice deficient in various genes involved in
DNA repair supported the idea that defects in DNA repair pathways are particularly
critical just prior to or during gastrulation. Mice homozygous for such mutations were
generally found to die around that critical stage. However, research on the influence
of an heterozygous state on embryonic survival and normality after exposure to low
doses of irradiation during this period is still at the beginning. In our laboratory, we
are investigating the influence of mutations in genes involved in various DNA repair
pathways (G1 double strand break repair by non homologous end joining, S phase
double strand break repair by homologous recombination, single strand break repair)
on the radiation sensitivity of early gastrulas. Results obtained todate showed a clear
increase of radiation-induced chromosome aberrations in the cells of various mutants,
compared to cells of wild-type (= “normal”) gastrulas given the same dose of X-rays.
In a next step, we will investigate whether this increased genetic sensitivity is
accompanied by an increase of radiation-induced embryonic malformations.
It should not be surprising that such genes, whose mutations lead to an increased
probability to develop cancer due to their important role in cell cycle control and
DNA repair processes, also determine to a large extent the sensibility of the embryo to
various developmental effects.
5. Summary and conclusions
The potential early loss of an (unsuspected) embryo is usually considered as of minor
importance, compared to the risk of a newborn being malformed. This clearly
explains why, most often, no particular precautions are taken to avoid unnecessary
exposure of a preimplantation embryo in women not aware to be pregnant. However,
various results obtained during recent years have suggested that, like a number of
chemicals, ionizing radiation could be potentially teratogenic in a few mouse strains,
when administered as early as at the one-cell (or "zygotic") stage. The Japanese and
German results also suggested that irradiation of later preimplantation stages could
induce similar effects, though to a lesser degree. While the German studies have
clearly shown that, in the Heiligenberger strain, a genetic predisposition exists for
induction of gastroschisis, this is not necessarily so for the various malformations
induced in other mouse strains.
Irradiation of early preimplantation embryos could also induce a genomic instability
in the surviving foetuses. Such effect could be of a general nature but would occur
more frequently in malformed foetuses. Its potential consequences remain to be fully
determined.
Mutations in genes concerned with cell cycle regulation, apoptosis or DNA repair
could increase the radiation sensitivity of the embryo. When present at the
homozygous state, such mutations revealed most often lethal, leading to embryonic
mortality soon before or after implantation. Research on the incidence of
heterozygous mutations on the radiation sensitivity of the early embryo is still scarse.
Even if embryonic lethality seems to represent, by far, the principal risk associated
with an exposure of the very early embryo to ionizing radiation, the above-reported
results could be of some concern for radiation protection. This is the reason why a
scientific seminar was recently organized by the European Commission in
cooperation with the Group of experts referred to in Article 31 of the Euratom. The
discussion which followed led to the following conclusions and recommandations :
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Current regulations (the European Basic Safety Standards Directive) are
characterized by a prudent approach and ought not to be changed on the basis of
the new observations concerning the effects of irradiation during the first trimester
of pregnancy. Nevertheless, these new observations have various practical
implications, particularly in the medical field and in intervention situations :
-
Due to genetic factors, there could be for some individuals a higher risk of
radiation-induced malformations or lower thesholds;
-
Even in the absence of genetic factors, irradiation during the preimplantation
period (when women are not aware of being pregnant) could induce congenital
malformations or genomic instability (above some threshold dose);
-
This requires cautiousness in the medical field; the application of the ten-day
rule (plan the non-urgent examination within the ten days following the
beginning of the menstruation), whenever the abdominal dose could be
significant, would largely reduce these problems
It was also concluded that more research is needed in this field, particularly as regards
heterozygotes for genes implicated in DNA check-point integrity and DNA repair, as
well as induction of genomic instability. Such research is under way in our laboratory,
with particular emphasis on the potential relationships between developmental effects,
genomic instability and changes in gene expression in embryos from radiosensitive
strains irradiated during the very early stages of pregnancy.
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