J. Radiat. Res., 47: Suppl., B83–B97 (2006)
Transgenerational Effects of Radiation and Chemicals
in Mice and Humans1
Taisei NOMURA*
Transgenerational effects/Radiation/Chemicals/Mice/Humans.
Parental exposure of mice to radiation and chemicals causes a variety of adverse effects (e.g., tumors,
congenital malformations and embryonic deaths) in the progeny and the tumor-susceptibility phenotype is
transmissible beyond the first post-radiation generation. The induced rates of tumors were 100-fold higher
than those known for mouse specific locus mutations. There were clear strain differences in the types of
naturally-occurring and induced tumors and most of the latter were malignant. Another important finding
was that germ-line exposure elicited very weak tumorigenic responses, but caused persistent hypersensitivity in the offspring for the subsequent development of cancer by the postnatal environment.
Activations of oncogenes, ras, mos, abl, etc. and mutations in tumor suppressor genes such as p53
were also detected in specific tumors in cancer-prone descendants. However, the majority of tumors
observed in the progeny were those commonly observed in the strains that were used and oncogene activations were rarely observed in these tumors. It can be hypothesized that genetic instability modifies
tumor occurrence in a transgenerational manner, but so far no links could be established between chromosomal and molecular changes and transmissible tumor risks. Our data are consistent with the hypothesis
that cumulative changes in many normal but cancer-related genes affecting immunological, biochemical
and physiological functions may slightly elevate the incidence of tumors or fasten the tumor development.
This hypothesis is supported by our GeneChip analyses which showed suppression and/or over-expression
of many such genes in the offspring of mice exposed to radiation.
In humans, a higher risk of leukemia and birth defects has been reported in the children of fathers
who had been exposed to radionuclides in the nuclear reprocessing plants and to diagnostic radiation.
These findings have not been supported in the children of atomic bomb survivors in Hiroshima and
Nagasaki, who were exposed to higher doses of atomic radiation. However, it will be important to follow
the human subjects, especially for adult type cancers and chronic diseases throughout their lives to determine whether the mouse studies can predict human responses.
“To a Mouse”
On turning her up in her Nest, with the Plough,
November, 1785
I’m truly sorry Man’s dominion
Has broken Nature’s social union,
An’ justifies th’ ill opinion,
Which makes thee startle,
At me, thy poor, earth-born companion,
An’ fellow-mortal!
*Corresponding author: Phone: +81-6-6879-3810,
Fax: +81-6-6879-3819,
E-mail: [email protected]
Department of Radiation Biology and Medical Genetics, Graduate School
of Medicine, Osaka University, Suita, Osaka 565-0871, Japan.
1
Special lecture
The best laid schemes o’ Mice an’ Men
Gang aft a-gley,
An’ lea’e us nought but grief an’ pain,
For promis’d joy!
An’ forward, tho’ I canna see,
I guess an’ fear!
(Robert Burns, November, 1785)
[The above is an extract from a poem entitled “To a mouse”
written in 1785, the era of industrial revolution, by Robert
Burns; in which he expressed concern for the health of mice
and humans owing to the destruction of their environments
by humans themselves or by human oppression. It was written on the occasion of his turning up the nest of the mouse,
which was at that time considered to be the smallest and
weakest mammal and which had prepared for and expected
a comfortable life for the coming winter. This view was ech-
J. Radiat. Res., Vol. 47, Suppl. B (2006); http://jrr.jstage.jst.go.jp
B84
T. Nomura
oed much later in the essay (Nomura, Nature, 1990) entitled
“Of mice and men?”. It considered, in the context of mouse
experiments, concerns for our future generations in an era of
advanced science and energy revolution.]
INTRODUCTION
Cancer is among the leading causes of death in childhood
in the developed countries of the world. In our investigations
reported in this paper, cancers were induced in mice by ionizing radiation or chemicals either by in utero or germ cell
exposures. In addition to cancers seen in the progeny, there
were other adverse effects, e.g., abortions and congenital
malformations. Extensive studies on in utero exposures were
conducted from late 1960’s to mid-1970’s, and 41 chemicals
were proven to induce cancer in the offspring.1) These studies had been referred to under the heading “Transplacental
Carcinogenesis”, in view of the fact that placental transfer
of the chemical carcinogen to the fetus resulted in cancers
detected after birth. When chemical carcinogens such as urethane, dimethylbenz(a)anthracene, ethylnitrosourea, diethylstilbestrol etc. were administered to pregnant animals after
the period of organogenesis, tumors developed in various
organs; however, malformations were induced at the organogenesis stage.2–9) These mouse data were supported by
human studies, which showed that vaginal cancers developed in young women whose mothers had been treated with
diethylstilbestrol during pregnancy for threatened abortions.10) A higher risk of cancer was also observed among
children exposed to atomic bomb radiation in utero.11) Other
adverse effects of in utero exposure include those on gonadal
development and organ function6,12) and hypersensitivity of
the organ for the future development of tumors by postnatal
environmental factors.13,14)
Although it is expected that exposure of germ cells to
radiation or chemical mutagens will cause adverse effects
such as cancer, malformation, abortions, etc. in the offspring, only a very limited number of studies had been
focused on this question and on the potential causal mechanisms. In order to fill this gap in knowledge, in 1967, we
launched the first and largest series of mouse experiments
with the ICR strain and in 1981 extended them to N5 and
other strains. We found that radiation and chemicals induced
germ cell alterations causing tumors, malformations and
embryonic deaths in the offspring.1,15–24) These studies had
been referred to under the headings of “Transgenerational
Carcinogenesis and Teratogenesis”, “Paternal Toxicology”,
or “Male-mediated Developmental Toxicology”,20,23,25,26)
although preconceptional exposure of females also induced
such effects in the offspring.15–18)
In humans, a higher risk of leukemia and congenital malformations has been reported in the children of fathers who
had been exposed to radionuclides in the nuclear reprocessing plants and to diagnostic radiations.27–33) However, no
increases in adverse effects (mutations, malformation, cancer, etc.) have been demonstrated in the children of atomic
bomb survivors in Hiroshima and Nagasaki, who had been
exposed to higher doses of atomic radiations.34)
In this article, I first present an overview of the results of
mouse studies carried out in our laboratory and then discuss
our subsequent molecular studies aimed at gaining insights
into the possible mechanisms underlying transgenerational
effects of radiation and chemicals. Using these as a framework, I consider the question of transmissible genetic risk
and our future.
EMBRYONIC DEATHS AND CONGENITAL
MALFORMATIONS
The initial X-ray experiments were carried out with ICR
mice most extensively from 1969 to 1975 and the data were
analysed subsequently (1975 to 1981). Adult male mice
were exposed to radiation and then mated with untreated
estrous females at various times before conception; vaginal
plugs were checked to determine the date of conception and
the stages of germ cells exposed. Some mice were euthanized on the 18th day of gestation and F1 fetuses were examined for embryonic deaths, congenital malformations, etc.
by Caesarian operation.15–17) Other parental mice delivered
live offspring, which were treated postnatally with tumorpromoting agents (with appropriate concurrent controls) and
screened for congenital malformations, tumors, chronic diseases, and related chromosomal and molecular changes.1,15–
24, 35–37)
Figure 1 summarizes data showing that paternal exposure
to radiation induces dominant lethality in the offspring.
Exposure of males to radiation and some chemical mutagens
at postmeiotic stages resulted in a high incidence of dominant lethals. The frequencies increased with increasing doses
of X-rays, e.g., about 60 percent dominant lethality after an
X-ray dose of 5 Gy to spermatids.16,17) Irradiation of female
mice also caused a high rate of embryonic deaths. The estimated rates of induction (per Gy) were 6.7 × 10–2, 10.9 ×
10–2 and 6.5 × 10–2/Gy for spermatozoa, spermatids and
oocytes at late follicular stages, respectively.16,17,20,23) Protracted irradiation of post-meiotic stages did not result in any
reduction in the frequencies of dominant lethals. Spermatogonial exposures, however, hardly ever induced dominant lethals, because dominant lethals are caused by large
chromosomal aberrations and most spermatogonia with such
aberrations are killed or eliminated before or during meiosis.16,17,20,23) The inference from these mouse results is that
in the F1 of atomic bomb survivors, no significant increases
in abortions would have been expected, because germ cells
sampled in most of the radiation-exposed male parents were
irradiated as spermatogonia.
Returning now to the mouse studies, in the surviving
fetuses, various types of congenital malformations were
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Transgenerational Carcinogenesis
observed (Fig. 2).15–17,20) Among these were: cleft palate,
hydrocephalus, gastroschisis, buphthalmus, diaphragm hernia, dwarfism, tail anomalies, etc. For defining dwarfism,
B85
a consequence of intrauterine growth retardation, the
criterion15,16) was that the body weight of the fetus was less
than 75% of that of the rest of the litter. Most of the induced
Fig. 1. Embryonic deaths (dominant lethals) induced in F1 fetuses of mice exposed to ionizing radiation16,17–20). Incidence
of dominant lethals (% of dead embryos per implant) was shown after acute doses of X-rays to male mice at spermatozoa,
spermatids and spermatogonia stages. Similar results were obtained in the offspring after female exposure (see details in
the text).
Fig. 2. Morphological anomalies found in the progeny of mice exposed to radiation and chemicals. a; buphthalmus, b;
general edema, c; oligodactyly, syndactyly (2nd and 3rd fingers) and polydactyly (first toe) (left to right), d; exencephaly
and open eyelid, e; dwarf and open eyelid, f; hydrocephaly and cleft palate (left) (right; normal), g; gastroschisis, h; tail
anomaly and meningocele, i; hydatidiform mole, j; defect of right testis and epidydymis, k; diaphragm hernia.
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B86
T. Nomura
malformations are those that are also commonly observed in
humans, except tail anomalies. However, tail anomalies correspond to the abnormalities of vertebral bones in humans.
Similar results were obtained in experiments in which the
females were irradiated.15–17) The rates of induction of congenital malformations in the F1 fetuses (all per Gy) were
11.6 × 10–3, 6.6 × 10–3, and 10.4 × 10–3 , respectively for
post-meiotic, spermatogonial and oocyte exposures.16,17,20,23)
Post-meiotic stages were twice as sensitive as spermatogonia.
In addition to the scoring of congenital malformations
prenatally, (i.e., through in utero analyses of pregnant
females euthanized on day 18 of gestation), live born progeny were examined 7 days after birth to ascertain those malformations compatible with viability.1,15–17,20) Induced rates
per Gy were 4.5, 1.9, and 6.2 × 10–3, respectively, for irradiated post-meiotic, spermatogonial and mature oocyte stages.16,17,20,23) In general, the proportion of congenital malformations detected prenatally was 2–3 fold higher than that
detected after birth (anomalies such as cleft palate, exencephaly, gastroschisis, buphthalmus and some dwarfs were
lethal shortly after birth), indicating that about 70% of the
malformations in mice were lethal shortly after birth (in
humans, neonates with cleft palates, gastroschisis, etc., survive after surgical procedures).
Some viable malformations, such as dwarfism, open eyelids and tail anomalies were transmitted to further generations with varying degrees of expressivity.15,16,17,20,23) Supporting evidence for the X-ray induction of congenital
malformations in mice and for their transmission have been
published by Kirk and Lyon38,39) and Lyon and Renshaw40)
using the same doses, treated stages and criteria as in our
studies16) but with a different strain. Searle and Beechey41)
showed that the phenotype of growth retardation is transmissible as an autosomal dominant albeit with variable penetrance. Müller et al.42) showed that in the mouse strain
‘Heiligenberger Stamm’ there was an increase in the frequency (4.5% vs 2.3% in the control) of one particular malformation, namely gastroschisis in 19-day-old fetuses after
137
Cs γ-irradiation of meiotic stages of parental males
(reviewed by Streffer, C. in this volume). In this strain, however, this malformation is known to occur at a high frequency in controls (around 2% in some experiments and up to 4%
in others).
The induced rate of congenital malformations in our
experiments was slightly higher than that of dominant skeletal mutations,43) because we scored more abnormalities.
Spermatogonial exposure induced a 0.7% increase of malformed fetuses per Gy, and only 0.2% increase per Gy in the
offspring scored 7 days after birth.15–18,20,23) Incorporating
these values, one can estimate that in the children of atomic
bomb survivors (mean parental dose of about 0.42 Sv) only
a very small increase (i.e., 0.3% increase of malformed
fetuses) would have been expected. Since the baseline fre-
quency of congenital malformations was about 5% in Japanese populations, such a very small increase in the children
of atomic bomb survivors would be undetectable. In fact,
frequency of untoward pregnancy outcome (malformation,
stillbirth, and prenatal death) is 5.0% in control (< 0.01 Sv)
and 5.2% in exposed (> 0.01 Sv) group44) (reviewed by
Nakamura, N in this volume).
These studies were extended to a variety of chemicals.15–
17,20,23,45)
It is of interest to note here that treatment of males
with N-ethyl-N-nitrosourea (ENU), the most potent chemical mutagen known to date,46) resulted in progeny with
asphyxia (respiratory distress syndrome; RDS), in addition
to morphological anomalies (malformations).47) After a caesarean operation on day 18 of gestation, most fetuses can
breath and survive by resuscitation. However, a few fetuses
do not breathe and die as it happens in human neonates
(asphyxia).
In our experiments,47) the incidence of fetuses showing
RDS and morphological anomalies was higher after spermatogonial than after post-meiotic germ cell exposure. This
finding is reminiscent of that of Russell et al. for ENUinduced specific locus mutations in male mice.46,48,49) Since
ENU shows low killing effects, ENU-treated spermatogonia
would be expected to survive during meiosis and cause a
high frequency of mutations and also malformations in F1
offspring. These ENU studies were carried out in the 1980s,
and at present, ENU saturation mutagenesis is a useful method to produce various mouse models for human diseases.
Worthy of note is that RDS was also inducible by radiation. However, unlike in the case of ENU, the frequencies
were higher after irradiation of post-meiotic germ cells than
of spermatogonial cells (as has been known for radiationinduced specific locus mutations.49)). The rate of RDS is also
higher than that of morphological malformations (Fig. 3).
Other human data
In humans, epidemiological studies suggested a significantly higher incidence of congenital malformations in the
offspring of North Vietnamese soldiers who had fought in
the mountain area and were exposed to herbicides containing dioxin (and married to unexposed women in North Vietnam) in comparison with those of north Vietnam soldiers
who did not engage in the war in South Vietnam or fought
in the coastal area (not exposed to dioxin).50,51) The report
of the U. S. National Academy of Sciences noted that there
was suggestive but not definitive evidence for a higher incidence of congenital malformations and also some cancers in
the children of U. S soldiers who fought in the Vietnam
War.52)
A significant increase of stillbirths and specific congenital
malformations was reported in the offspring of fathers who
were exposed to radiation at the Sellafield nuclear reprocessing plant (Odds ratios of 1.21 to 1.69 at 100 mSv).33) Relative risk values for stillbirths and congenital malformations
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Transgenerational Carcinogenesis
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Fig. 3. Congenital malformations and respiratory distress syndrome (RSD) in the F1 fetuses of male N5 mice
exposed to 5.04 Gy of X-rays. The numbers above the bars are total numbers of fetuses and numbers in parentheses
are numbers of their dam. Sp-zoa; Spermatozoa and spermatid (postmeiotic) stages, Sp-gonia; spermatogonia
were significantly higher after post-spermatogonial exposure, but very low after spermatogonial exposure. It was also
the case for the induced rate of congenital malformations in
mouse experiments.1,16,17,20,23) However, there are large differences in the radiation doses between human and mouse
studies,1,17,20,53) as it was also in the leukemia study.22,30)
TUMORS IN THE OFFSPRING
It is a well-known fact that the incidence of cancers is
influenced and/or modified by postnatal environments. Consequently, the investigation has to be carried out using high
quality animal and animal facilities and a strict coding system.1,36) In our experimental series, the mouse numbers were
expanded and randomly selected animals were exposed to
radiation or chemicals,15–23) and unexposed litter mates were
used for the concurrent controls. Live offspring of preconceptionally exposed or unexposed parental mice were nursed
in the barrier (SPF) section with or without postnatal treatments, and the presence or absence of tumors was determined macroscopically and microscopically at autopsy. All
procedures between treatment and autopsy were kept blind.
After the autopsy, F1 offspring were matched to their parents
retrospectively by their code number, and classified according to the irradiated or unirradiated status of the parents.
Their descendants (F2, F3, etc.) were also classified as to the
retrospectively determined tumor-phenotype of their parental (F1, F2, etc.) mice. Fig. 4 shows one of the general
scheme that was followed.1,36)
Dose responses and stage sensitivities
Initial studies were carried out with ICR mice using Xrays (dose rate; 0.72 Gy/min).15–18) Male ICR mice were
exposed to a single or fractionated (protracted) doses (0.36
Gy of X-rays given 2 hours apart) and germ cells exposed
as spermatozoa, spermatids and spermatogonia were sampled.16,17) Females were also exposed to X-rays at various
intervals. Eighty-seven per cent of the induced tumors in
ICR mice were found in the lung, the remainder being of
various types – ovarian tumors, lymphocytic leukaemia,
stomach tumors, lipoma, granulosa cell tumors, thyroid
tumors, liver haemangioma and hepatoma.16,17) The spectrum of tumor incidence in the offspring of treated animals
is essentially the same as that in controls (90% in the lung).
The incidence of tumors in F1 offspring increased with Xray doses to the parents in the range from 0.36 to 5.04 Gy.
Induced frequencies of lung tumors per Gy were 2.27, 1.49
and 3.17 × 10–2 for irradiation of postmeiotic stages (spermatozoa and spermatids), spermatogonia, and oocytes,
respectively (Note that the values for male germ cell stages
were obtained by averaging the rates at doses of 2.16 and
5.04 Gy; those for oocytes represent the average of the rates
at doses of 2.16, 3.6 and 5.04 Gy.15–17,35)). Frequencies (per
Gy) by protracted irradiation were 2.09 (postmeiotic male
germ cells), 0.09 (spermatogonia) and 0.29 × 10–2 (oocytes).15–17,35) It is evident that post-meiotic stages are more
sensitive than the spermatogonial stages. No differences
were observed in the incidence of tumors between single and
protracted doses after post-meiotic exposure.16,17) However,
with fractionated doses given to spermatogonia, large reductions in tumor incidence in F1 offspring were observed, the
figures approaching those seen in controls.16,17) Oocytes in
the mature follicle stages were resistant to single dose of Xrays up to 1 Gy for tumor induction in F1, but large increases
were observed at higher doses.16,17) Again, dose fractionation
to mature oocytes showed a large reduction in tumor
incidence16,17) as was also the case with fractionated spermatogonial irradiation. These observations suggest signifi-
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B88
T. Nomura
Fig. 4. Tumors and congenital anomalies in the offspring of male N5 mice exposed to 2.16 Gy of X-rays at the spermatogonial stage. This is one of the pedigrees of tumor-susceptible offspring of N5 mice exposed to X-rays. A part of the
pedigree of concurrent (untreated) controls is shown on the right side. Hepatoma lesions and/or normal liver tissues of 6
circled male offspring in the figure were used for GeneChip analysis. This figure was reproduced from T. Nomura et. al.
Cytogen. Genome Res., 200337) with permission. Closed symbols for males and females indicate tumor-bearing offspring,
and dotted symbols indicate offspring with congenital anomalies. Open symbols indicate offspring without tumors and
anomalies. The abreviations used are: Hep, hepatoma; LT, lung tumor; OC, ovarian tumor; LL, lymphocytic leukemia;
ScT, subcutaneous tumors (fibrosarcoma, rhabdomyosarcoma); LiT, liver tumor; RS, reticulum cell neoplasia; AH, atresia
hymenalis; D, dwarf; T, tail anomalies; SD, diverticulum of stomach.
cant repair capabilities of spermatogonia and mature
oocytes. Later, the results were confirmed with low dose rate
exposure to 60Co γ-rays at the spermatogonial stage.1)
The germ cell sensitivity for tumor induction in the offspring is very similar to that of specific locus mutations
induced by radiation,48,54–57) but the tumor frequencies are
more than 2 orders of magnitude higher than that of specific
locus mutations. Consequently, in order to examine the generality of our observations, studies were carried out with two
other inbred strains of mice (LT and N5).19) F1 progeny of
LT males irradiated at post-meiotic stages showed significantly higher frequencies of lung tumors and lymphocytic
leukemia (Table 1). Induced incidence of lung tumors in LT
was similar to that in ICR mice.16,17,19) However, much higher induced incidence of leukemia was observed in LT than
in ICR.
Post-meiotic treatment of the N5 males induced very high
incidences of tumors.19) Spermatogonial treatment of N5
mice increased the frequencies of various tumors such as
lung tumors, ovarian tumors, leukaemia, hepatoma, multiple
embryonic tumors, etc. in the F1 offspring. Induced rates of
lung and ovarian tumors in N519) were similar to those in
ICR.16) However, N5 mice showed higher incidences of lym-
phocytic leukemia and hepatoma than ICR and/or LT. Consequently, there are clear strain differences in the incidence
of lymphocytic leukemia and hepatoma among these 3
strains (Table 1).19,36)
Heritable nature of tumors
To confirm that the induced tumors are heritable, the F1
progeny of treated parents were mated and their progeny
examined.16,17) The ICR mice were mated as young adults
and the presence or absence of tumors was determined at 8
months of age. The progeny were then classified as to the
retrospectively determined tumor-type of the parent. Significantly higher frequencies of lung tumors were observed in
the F2, when their parental F1 had tumors. In each case the
original treated mouse was a male, as a precaution against
the possible transmission of the chemical or cytoplasmic
factors to the progeny. The results were confirmed by continuing the X-ray treated group into the F3 generation (by
mating F2 males with unirradiated females).17) Again, lung
tumor incidence was significantly higher in the F3, when F2
male parent had tumors. The pattern of inheritance is that of
a dominant with about 40% penetrance. Reduced penetrance
was also found for dominant skeletal mutations.43,48) Similar
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Transgenerational Carcinogenesis
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Table 1. Induced rate (× 103) of tumors in the offspring per parental dose (Gy) of radiation in different strains of mice
Strain
Radiation
TBA
Hepatoma
Leukemia and
lymphoma
Lung tumor
Reference
ICR
X-rays
24.6
0
1.9
22.7
16, 17
N5
X-rays
30.0
5.8
3.0
21.5
a
LT
X-rays
24.3
0
8.5
21.0
19
12.0
6.7
6.4
1.0
b
B6-C3F1
60
B6-C3F1
252
Co
Cf
–
800
–
–
63, 64
C3H/HeH
X-rays
–
–
–
0
66
BALB/cJ
X-rays
–
–
–
0
65
X-rays
–
–
6.8
–
68
H β-rays
–
–
13.2
–
68
–
90.0
65.0
22.5
61
N5
N5
CBA/JNCrj
3
X-rays
36)
From T. Nomura, Mutat. Res., 2003 with permission from Elsevier. TBA; tumor-bearing animal.
a
Spermatogonial exposure. Average of 2 doses (5.04 and 2.16 Gy).
Others are post-meiotic exposure.
b
Kamiya, K., Personal communication.
results were obtained with ICR males treated with urethane
and 4-nitroquinoline 1-oxide (4-NQO).16,17)
Inheritance of induced tumors was also examined in the
N5 strain,1,19,36) which develops various types of tumors.
Mating of preconceptionally-irradiated lung-tumor-bearing
Fl progeny with their litter mates yields a variety of tumors,
such as lung tumors, ovarian tumors, multiple embryonic
tumors, myxoma and thymic lymphomas (lymphocytic leukemias), etc. and congenital anomalies in the progeny of the
F2 generation. Observation continued for further generations. F3 progeny of F2 parental mice with multiple tumors
and lung tumors developed not only multiple tumors and
lung tumors, but also those of other types (ovarian tumor,
hepatoma, leukemia, etc), cataracts, and congenital anomalies (Fig. 4).1,36) Such a tendency has been found to persist
(up to F34; March, 2005). The pattern is similar to that of LiFraumeni syndrome in humans, suggesting the inheritance
of tumor susceptibility. We interpret these findings as indicating that germ cell exposure may have induced transmissible hyper-susceptibility to tumor induction in the next generation.
Cancer-prone progeny were used for chromosomal and
molecular analyses and these studies are considered later in
the paper.
Manifestation of germ-line alteration causing tumors
by postnatal environments
If germ-line mutation can lead to cancer in a given organ,
all cells composing that organ must be mutated and have an
equal likelihood to form tumors. However, only one tumor
nodule was induced in the organ. One can hypothesize that
the ‘mutational alteration’ transmitted from the irradiated
parent to the offspring is presumably weakly carcinogenic
by itself, and that its expression can be influenced by aging,
naturally existing carcinogenic and promoting agents in the
diet and environment.
The above hypothesis was proven by the findings that
unusually large clusters of tumor nodules developed in the
lung after postnatal treatment with small amounts of urethane (Fig. 5).18) Further supporting evidence was reported
for lung tumors in the offspring of paternally irradiated outbred SHR mice postnatally-treated with urethane,58) for skin
tumors by postnatal treatment with 12-0-tetradecanoylphorbol-13-acetate (TPA),59) and for leukemia by preconceptional 239Pu irradiation and postnatal methylnitrosourea treatment.60) However, such enhancing effects were not observed
when CBA/J male mice were irradiated and their offspring
were postnatally treated with urethane.61)
In order to confirm the postulated persistent hypersensitivity induced by exposure of germ cells to radiation, N5
male mice were treated with 2.16 Gy of 60Co γ-rays (0.52
Gy/min) at the spermatogonial stage and mated with untreated N5 females. Offspring (6 weeks old) were treated twice
a week with TPA for 18 weeks. Under these conditions, a
significant increase of skin cancer and leukemia was
observed in the offspring. However, no increases of skin
cancer were observed without postnatal TPA treatment.
Higher frequencies of skin cancer developed in F2 and further generations.1) The main conclusion that can be drawn
from these studies is that germ cell exposure to radiation,
although very weakly carcinogenic by itself, presumably
induces transmissible hypersensitivity to tumor induction in
the offspring.18) This is a starting point for the study of radiation induced genomic instability in germline,62) which may
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T. Nomura
Fig. 5. Distribution of lung tumor nodules induced by the postnatal treatment of urethane (0.45 mg/g of body weight at 21
days after birth) to the F1 offspring of male mice exposed (A) and unexposed (B) to 2.16 Gy of X-rays.18) Details are given
in the text. Similar results were observed in the offspring of female mice exposed to X-rays.18)
cause tumors in the next generation.
Further mouse studies
After the publication of Gardner’s report in 1990 on the
higher risk of leukemia in the children of fathers who had
been exposed to nuclear radiations,30) experimental studies
were carried out with various strains of mice in several countries (Table 1),1,24) to reconcile the differences between
mouse and human studies. A high incidence of hepatomas
was observed in the Fl offspring of C3H male mice exposed
to 0.5 Gy of 252Cf (66% neutron) and mated with C57BL/6
females.63,64) A small increase in tumor frequency was also
observed in B6C3Fl offspring after paternal exposure to 60Co
γ-rays (Kamiya, K., personal communication). Cattanach et
al.,65,66) however, observed no significant increases but
recorded a seasonal change in the incidence of lung tumors
in the offspring of BALB/cJ or C3H/HeH male mice
exposed to X-rays following the experimental protocol of
Nomura.16,17) Evidence for such seasonal changes in tumor
incidence has been published and this relates to experiments
carried out in insufficiently controlled animal facilities and
experimental conditions, e.g., a leak of outdoor light or
change of light-dark intervals significantly influenced tumor
frequencies in mice.67)
A Canadian group carried out experiments with N5 mice
provided by Nomura. In these experiments, male N5 mice
were irradiated with 5 Gy of X-rays under conditions close
to those of Nomura.17,19) The results showed that the probability of dying from leukemia and overall survival were statistically different between the offspring of X-ray-treated
males and of unirradiated controls. Earlier occurrence of
leukemia was also observed in the F1 offspring after the
treatment of male N5 mice with tritiated water.68)
A lifespan experiment showed a trend towards a higher
incidence of tumors of the hematopoietic system and broncho-alveolar adenocarcinomas in the offspring of male
CBA/JNCrj mice exposed to X-rays one week before mating
(spermatozoal exposure). However, no increase in tumor
incidence was observed in the offspring of males irradiated
9 weeks before conception (spermatogonial stage exposure).61)
In general, there are clear strain differences in the types
of induced tumors and tumor incidences in the preconceptionally irradiated offspring (Table 1), indicating that preexisting genetic predisposition i.e., strain differences, is a
critical component also in transgenerational radiation carcinogenesis. In other words, preconceptional irradiation may
increase background incidence of the tumors in each strain.
In fact, most of induced tumors were commonly observed
types in each strain, and only a few were of very rare types.
Malignancy of induced tumors and minor epigenetic
factors
A major thrust of our initial mouse studies15–17) was on the
malignancy of induced tumors, especially lung tumors. To
examine malignancy, we tested transplantability of induced
tumors in the offspring of X-irradiated N5 mice.19) Twenty
six tumors induced in N5 progeny (11 lung tumors, 5 leukemias, 2 fibrosarcomas, 3 undifferentiated tumors, 2 hepatomas, etc), were transplanted subcutaneously in N5 mice.
J. Radiat. Res., Vol. 47, Suppl. B (2006); http://jrr.jstage.jst.go.jp
Transgenerational Carcinogenesis
Among them, only 3 (1 lung tumor and 2 hepatomas) failed
to grow, but the others grew rapidly, metastasized and killed
the recipient N5 mice. Thus, mouse lung tumor is classified
as malignant.69) It grows gradually and the pathological features changes with elapsed time.5) Even very small lung
tumor nodules (1–2 mm diameter) can grow and kill the animals.5,19)
It was argued in 1990 on the occasion of Gardner’s report
22,30)
and recently (after the symposium) by Selby et al70) that
heavier mice are more likely to develop spontaneous and
induced tumors earlier and that, because of induced dominant lethality (i.e., decrease of litter-size), experimental
groups derived from radiation exposure of male germ cells
at postmeiotic stages contain heavier mice or rats. Accordingly, these animals develop more spontaneous tumors at a
faster rate than the control groups. The authors conclude that
the increased tumor rates have nothing to do with induction
of dominant tumor mutations.
The above argument and conclusion, however, is not a
valid one. It has long been known that the number of tumor
nodules is proportional to the size of the organ (i.e., the number of target cells) in given strains at given ages irrespective
of treatment (see, for instance, ref. 5). However, the differences are in the range of 5 to 15% of the incidence, depending on the litter size or body weight, but not 2 to 3-fold, as
is actually seen in tumor frequencies in the offspring
descended from germ cells exposed in the post-meiotic stages. Furthermore, as noted earlier, very little dominant lethality is induced after spermatogonial exposure and yet significant increases were found in tumor frequencies in the
offspring descended from irradiated spermatogonia. The situation is similar to that after paternal treatment with chemicals such as ENU, urethane, etc. It is therefore worth stressing that litter-size effects on carcinogenesis are at the level
of a few per cent, whereas strain differences on carcinogenicity differ by orders of magnitude. For example, the spontaneous incidence of lung tumors in A/J strain is 50% (34/
68), but it is 0% (0/250) in PT strain at the age of 12 month.
Multiplicity of tumor nodules in the lung is 500-fold different between two strains, e.g., 31.9 tumors per lung in A/J
(1338 tumor nodules per 42 mice) but 0.06 in PT (4 tumors
per 63 mice) in experiments on subcutaneous injection of
urethane (1mg/g body weight). One major gene determining
lung tumor susceptibility has already been mapped, and
there are many minor genes (i. e., those with small effects)
as well (data not shown).
Radiation and chemical treatments also caused functional
damage and defective gonad development.6,12–14) Of note are
the findings that in utero treatment with either chemicals or
radiation reduced the activity of catabolic enzymes, causing
higher incidence of tumors by second postnatal challenge by
chemicals or by environmental factors.12–14) Moreover, radiation and chemical exposure after the organogenesis stage
induced serious damage on the development of male and
B91
female gonads, e.g., scarcity of the sperm and oocytes in the
testis and ovary,6) suggesting adverse effects in further generations.1,15) In fact, early studies on transgenerational carcinogenesis were started from mice and rats treated with
chemical carcinogens in utero ( see ref. 15). Recently, it has
been reported that in utero exposure to endocrine disruptors
induced such efects in the male germ line which are transmitted to F2 and F3 generations.71,72) Since the effects correlate with altered DNA methylation pattern in male germ line,
environmental factors may reprogram the germ line and promote various diseases.71) However, the mating of outbred or
closed colony animals (random bred with several strains)
used in this kind of studies and also in some of early investigations on transgenerational carcinogenesis may result in
the segregation of genes derived from different strains which
show different DNA methylation pattern, tumor susceptibility, enzyme activities, etc., having no relation to radiation
induced genetic effects.
POSSIBLE MECHANISMS: CYTOGENETIC
AND MOLECULAR STUDIES
Transgenerational Chromosomal Changes
We studied radiation- and urethane-induced translocations
cytogenetically in the germ cells of treated adult ICR males
as well as in the F1 male progeny sired by them to examine
whether there was any correlation between induced translocations and tumors in the F1 progeny.16) Translocation studies in the F1 males descended from post-meiotic germ cells
of irradiated (5.04 Gy) parental males showed that of a total
of 81 F1 males, 10 had characteristic translocation configurations. A further 3 had a deficit of post-meiotic germ cells
in the seminiferous tubules (histological analysis). In 70
concurrent control F1 males, there were no translocations.
The F1 males were also screened for the presence of tumors
and congenital anomalies. However, tumors occurred no
more frequently in the offspring with translocations than in
those without translocations, i.e., there is no correlation
between the induction of translocations and the occurrence
of tumors.16,37)
Then we examined whether any relationship could be discerned between visible chromosomal changes (in bone-marrow preparations using G and CQ-band analysis) in 36
tumor-bearing offspring in the N5 strain (from post-radiation
generations F1 to F5) and 53 irradiated but non-tumor-bearing controls.19) Again, these results did not provide any evidence for a cytogenetically detectable chromosomal abnormality in these animals.19,37) The above sets of data
considered together suggest that induced germ cell changes
causing tumors are not related to gross chromosomal changes detectable with the cytogenetic techniques employed.
However, they do not exclude the possibility that smaller
genetic changes may be involved.16,17,19) These findings
were supported by the fact that urethane, an intragenic
J. Radiat. Res., Vol. 47, Suppl. B (2006); http://jrr.jstage.jst.go.jp
B92
T. Nomura
mutagen,73,74) induces tumors, but neither translocations nor
dominant lethals in the offspring.16,17)
Cytogenetic analysis (G-banding) of the abnormal fetuses
with morphological and functional defects (derived from
experiments involving paternal radiation exposure (5.04
Gy)) showed that numerical chromosomal abnormalities
were present in the abnormal fetuses.20,23,37) However, it is
difficult to discern a cause-effect relationship between these
and congenital anomalies for the following reason: numerical chromosomal changes were found in 7–40% of the analyzed cells of each affected fetus in spite of the fact that the
fetuses were derived from radiation exposure to spermatogonial cells of the parents. The question of whether germ cell
exposures have caused instability manifest as numerical
chromosomal changes in the fetuses remains open. The limited cytogenetic data on viable congenital anomalies induced
in the progeny show that most cases of dwarfism and some
of tail abnormalities are transmissible to further generations
and in those fetuses, deletions, inversions, or trisomies were
found.37)
Oncogene activation
In order to determine the contribution of oncogenes to
multi-generation carcinogenesis, activation of 17 known
oncogenes was examined in DNA extracted from transplantable tumors induced in the offspring of X-irradiated N5 parents (undifferentiated multiple tumors, fibrosarcomas, lung
tumors, lymphocytic leukaemias, etc.).21) Ras, mos and/or
abl genes were amplified in two rare tumors (undifferentiated multiple tumors in Fig. 4) and lymphocytic leukaemia.21)
Furthermore, two of three tumor DNAs which did not
hybridize with any oncogenes so far tested had transforming
ability on cultured cells from golden hamster embryos,21)
and were later found to contain mos and cot oncogenes.75,76)
(Cot was isolated from human thyroid cancer tissue). These
were the first reports of detection of activated mos and cot
genes by transfection assay.75) P53 mutations were also
detected in a brain tumor (glioma) of X-irradiated N5 progeny (data not shown). Thus, known and new oncogenes were
activated in the tumors of the descendants of X-irradiated N5
parents. In commonly observed types of tumors, however,
we rarely found oncogene activation and p53 mutations.
Consequently, the majority of tumors induced in the progeny of irradiated parents were commonly observed types in
the respective strains of mice in which oncogene activations
were rarely detected, although germ-line alterations could
produce some rare types of tumors and molecular changes.
Genomic instability
There is a considerable literature on germ line mutations
induced at the mouse ESTR (expanded simple tandem
repeat) loci by both low and high LET (neutrons from 252Cf)
irradiation.62,77–83) In the work of Dubrova et al,62,77–-80) however, spermatogonial stages were found to be more sensitive
than postmeiotic stages and there were no dose rate effects
for these mutations induced in spermatogonia. Their results
are thus different from those of other investigators81,82) and
are also at variance with findings from studies with specific
locus mutations49,54–57) as well as our mouse studies.16,17)
In order to examine whether genomic instability induced
by irradiation of germ cells shows some association with
induced congenital anomalies and tumors, we studied the
induction of ESTR mutations at the Pc-3 locus in the F1
progeny of males treated with 2, 3, 7, 8-tetrachlorodibenzop-dioxin (TCDD) and radiation. The choice of TCDD was
dictated by earlier work in which paternal exposure to
TCDD had been found to induce congenital anomalies in
man51,52) and mice72) (reviewed by Ryo, H., Nakajima, H.,
Nomura, T. in this volume). The frequency of band shifts
(estimated through electrophoresis assay of PCR products)
was very high (19/72; 26.4%) in the B6C3F1 from the untreated controls. There was no statistical difference between the
frequencies in the untreated and treated B6C3F1 [derived
from C3H/HeJ male mice treated with TCDD at doses 50
ng/g body weight (16/62; 25.8%) and 100 ng/g (12/53;
22.6%) and mated with C57BL/6J females]. Oral administration of TCDD (100 ng/g body weight) to C.B17 male
mice also induced congenital anomalies. The incidence of
RDS in fetuses of the TCDD-treated group was 29.9%,
while it was 11.7% in solvent (corn oil) controls (P < 0.01
by a Chi-squared test with Yates’ correction). There are no
differences in the mean base length in the fetuses of the
TCDD group relative to the corn-oil-treated groups [analysis
of the PCR products of the Pc-3 locus by Gene Scan (ABI
PRISM 3100 Genetic Analyser; Applied Biosystems, Foster
City, CA, USA)].37,72) However, DNA of abnormal base
length (over the 99% confidence limit of controls) was found
in 7 of 127 F1 fetuses in the TCDD group (1 of 140 in controls). Furthermore, 3 out of 7 had congenital anomalies
(short tail, kinky tail and omphalocele).
In order to study the possible association of genomic
instability with induced tumors, experiments were carried
out in our laboratory using the offspring of 60Co-irradiated
(postmeiotic irradiation; 2.16 Gy; 0.57 Gy/min) and unirradiated N5 males. Mice were euthanized at twelve months of
age and liver and tumor tissues were examined for Pc-3
mutations. The mean base length of 162 offspring from unirradiated parental males was 489.00 ± 1.56 (99% CI) and, as
of now, there was no increase of outliers either in 65 offspring of irradiated males or in 13 tumor tissues. Further
studies are currently underway using microsatellite probes in
the offspring of γ-ray and neutron exposed male mice. The
preliminary results show that microsattelite mutations could
be induced by neutron in the offspring (data not shown),
though the increased rate of microsattelite mutations have
not yet been found in the children of Chernobyl liquidators
exposed to low doses (< 50 mSv) of radiations.84)
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Transgenerational Carcinogenesis
Changes in gene expression in cancer-prone progeny
As discussed earlier, the important features of the presumed germ-line alterations causing tumors in the progeny
of irradiated mice that we have observed are: (1) 100-fold
higher incidence of tumors in the offspring than ordinary
(i.e., specific locus) mouse mutations; (2) weak but transmissible carcinogenicity of radiation by itself with manifestation by postnatal environment; (3) inheritance of tumor susceptibility; (4) strain-dependent differences in background
and induced incidence of tumors. All these considered
together suggested to us early on 197816) that the accumulation of changes in normal functional genes involved in
immunological, biochemical, and physiological functions
may slightly elevate tumor incidences or fasten the tumor
development in each strain.
Since changes in the expression of many genes are known
to occur in tumors,85) we are now carrying out gene expression studies to test the above hypothesis using the Gene Chip
technology (Affymetrix Inc., Santa Clara, CA, USA). In
these experiments, the affected F1 offspring (with tumors or
malformations) of N5 male mice exposed to 2.16 Gy of Xrays at spermatogonial stage were used as the starting material. These animals were mated to their litter mates as young
adults and their offspring were examined for the presence or
absence of tumors at twelve months of age and classified as
to the retrospectively-determined type of the parent.
In one study using skin cancer material, among 6,500
genes examined in the F3 offspring of 60Co-γ-ray irradiated
male N5 mice, 254 and 75 functioning genes (in the skin
cancer lesion and the surrounding normal skin area, respectively) showed more than 4-fold differences in the expression level (both increases and decreases) compared to unirradiated concurrent control F3. In the non-tumor area, for
instance, macrophage inflammatory protein, osteopontin
B93
precursor, SV40 induced 24p3 mRNA, and a variety of
genes were 50-fold overexpressed or suppressed.1,36)
In similar studies comparing gene expression in hepatoma
and normal liver tissue of the affected offspring and controls
(Fig. 4), progeny No. 7 developed only one hepatoma. However, No. 13 developed 3 hepatoma nodules and both No. 35
and No. 43 developed 2 hepatoma nodules. Among 12,000
genes examined, more than 4-fold differences were observed
in 30 and 110 functioning genes in the normal liver tissue
and hepatomas, respectively, of irradiated F1 and F2 offspring (Table 2). The average number of oncogenes and
related genes with abnormal expression was 4.3 and 20.8 per
normal liver or hepatoma tissue, respectively. About 60% of
genes showing altered expression were different among 3
hepatoma nodules in No. 13 and 2 nodules in No. 35 and 43.
These suggest that each tumor nodule is derived from a different liver cell in the individual mouse. More precise analyses of the genes involved, tumor types, etc. are necessary,
before definitive conclusions can be reached.
In any case, it is clear that considerable numbers of genes
are abnormally expressed, such altered genes pre-exist in the
non-tumorous liver tissue of cancer-prone progeny, and the
majority of the abnormally expressed genes are those
involved in normal physiological, biochemical and immunological functions. Consequently, changes in gene expression
seem to occur in various normal functional genes rather
than oncogenes per se in irradiated cancer-prone or tumorsusceptible descendants, and their progressive accumulation
may contribute to cancer as we have hypothesized.16)
Knowledge of the numbers of such cancer-related genes and
tumor suppressor-related genes has been increasing enormously during the past few years with the advance of biomedical research and technology (the data of Net Aff Analysis Center (Affymetrix Inc.) (A, B in Table 2).36,37)
Table 2 . Changes in expression of functioning genes in the hepatoma and adjacent normal liver tissue of the descendants of N5
male mice exposed to 2.16 Gy of spermatogonial X-irradiation.
Increase ≥ × 4
Decrease ≤ × 1/4
Total
A
B
C
Total
A
B
C
12,000
1,888
390
725
12,000
1,888
390
725
Normal tissue
22.8
3
0.8
1.8
7
1.3
0
0.8
Hepatoma
83.9
16.4
3.3
6.1
26
4.4
0.1
3.6
Total numbers of genes
Average numbers of genes with abnormal expression
Eight hepatomas and 4 adjacent normal liver tissues of 4 male offspring of X-irradiated N5 male mice and normal liver tissues of 2
concurrent controls (Fig. 4) were used for Gene Chip analyses (U74Av.2 Array, Affymetrix, Inc., Santa Clara, CA, USA).
Gene expression in the hepatoma and normal liver tissues of the offspring of X-irradiated male N5 mice was compared to those of
concurrent controls (offspring of unirradiated N5 mice).
Numbers of functioning genes showing 4-fold increases or decreases were scored. The average values of 8 hepatomas and 4 normal
liver tissues are shown in the table.
A; oncogenes and oncogene-related genes, B; tumor suppressor genes and tumor suppressor-related genes,
C; immune-related genes. Data derived from NetAffx Analysis Center (Affymetrix Inc.) on July 29, 2005.
J. Radiat. Res., Vol. 47, Suppl. B (2006); http://jrr.jstage.jst.go.jp
B94
T. Nomura
HUMAN STUDIES AND PERSPECTIVES
In 1990, Gardner et al. reported that there was about 6–8
fold higher risk of leukemia (mostly acute lymphocytic leukaemia) in the children of fathers who were employed at
Sellafield nuclear reprocessing plant and had been exposed
to 10-100 mSv of radiation before conception.30–32,35) In particular, extremely high risk (about 7-fold) of childhood leukemia from the father’s exposure to doses as small as 10
mSv30) during the 6 months before conception suggested that
the postmeiotic sperm was more sensitive to radiation than
spermatogonia. The general pattern, e. g., germ cell stage
sensitivity, was very similar to that seen in our mouse
experiments16–19,22) and also in mutational sensitivity in mice
by specific loci method.49,54–57) However, it should be borne
in mind that in the human studies, induced rate of leukemia/
mSv was 3 to 150 fold higher (depending on the mouse
strains used) than in the mouse experiments,22,35) and
besides, mouse tumors did not show any increases in frequency in the progeny after protracted or chronic spermatogonial irradiation.1,16–19)
As a possible cause of leukemia induction, genetic damage induced in the sperm of fathers exposed to radiation was
proposed by the authors30) on the basis of our mouse experiments.16–19,22) However, this interpretation has not been supported by the epidemiological study on the children of atomic bomb survivors in Hiroshima and Nagasaki who were
exposed to an average dose of 435 mSv,34,86) although some
epidemiological studies reported (but have not proven) an
increase of leukemia in the children of fathers who had been
exposed to low doses of diagnostic radiation.27–29,32)
In part, the discrepancies between the findings in Hiroshima/Nagasaki studies and the Sellafield studies can be reconciled by considering the following.22,35) (1) Different germ
cell stages exposed in the two populations; Most of F1 offspring of atomic bomb survivors in Hiroshima/Nagasaki
were exposed to radiation at the spermatogonial stages
which shows reduced sensitivity to radiation compared with
sperm and spermatids. (2) Different germ cell susceptibility
to leukaemia induction may exist in Japanese and English
people as seen in race differences of lung cancer in white
and non-white uranium miners, though not by germinal
exposure, and also in large strain differences in mice. (3)
Postnatal tumor-promoting (radiation and/or chemically
contaminated) environment in Sellafield, as suggested by
our mouse experiments.18) Nevertheless, there is a surprisingly large difference in induced rate and doubling doses in
the mouse and Sellafield human studies.22,35) This disparity
could result from undetermined doses of neutron and internally incorporated radionuclides and chemicals and from
large variance in the risk value because of small sample size
in the Sellafield study.
Although a direct link between cancer and irradiation of
sperm has not been established in Sellafield study32) and cancer incidence from 1958 to 1997 did not increase in the children (below 20 years of age) and young adults (after 20;
median age at diagnosis 39.7 years) of atomic bomb survivors,86) continued surveillance of the children of exposed
populations is necessary for adult type cancers and other
adult (chronic) diseases which may increase at cancer-prone
ages, as seen in in utero atomic bomb exposed children. This
view gains support from our mouse experiments,1,17–19,
22,36,37)
because the induced rate of solid tumors in the offspring of mice exposed to radiation is much higher than that
of leukemia. In Japan, an epidemiological survey has been
running since 2002 to investigate the possibility of transgenerational transmission of chronic diseases in the offspring of
atomic bomb survivors, in addition to cancer study. Moreover, a collaborative study on microsatellite mutation is also
going on in Osaka and Hiroshima.
AKNOWLEDGEMENT
The studies reported in this article were supported by the
grants from MEXT of Japan and JSPS, Princess Takamatsu
Foundation, Nissan Science foundation, Nuclear Safety
Research Association, Showa-Shell Foundation, Heiwa
Nakajima Foundation, Japan Space Forum, and Amway
Japan, and Osaka Science Prize, Inoue Science Award, Kihara Memorial Awards, and Etoh Memorial Award. I wish to
thank all the past and/or present members of the First
Department of Surgery, former Institute for Cancer
Research, and Department of Radiation Biology and Medical Genetics, Graduate School of Medicine, Osaka University, and Medical Genetics, University of Wisconsin for their
advice and help over the past 30 years, Prof. J. F. Crow,
Madison, U.S.A. and Prof. K. Sankaranarayanan, Leiden,
The Netherlands for their constructive comments on this
manuscript and editorial help.
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Received on June 3, 2005
Final Revision Received on November 30, 2005
Accepted on December 3, 2005
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