Environmental signaling and evolutionary change: can exposure of
pregnant mammals to environmental estrogens lead to epigenetically
Carlos Guerrero-Bosagna,a,b,Ã Pablo Sabat,b,c and Luis Valladaresa
aLaboratorio de Hormonas y Receptores, Instituto de Nutricio´n y Tecnologı´a de los Alimentos (INTA), Universidad de Chile,Santiago, ChilebLaboratorio de Ecofisiologı´a Animal, Departamento de Ciencias Ecolo´gicas, Facultad de Ciencias, Universidad de Chile,Santiago, ChilecCenter for Advanced Studies in Ecology & Biodiversity and Departamento de Ecologı´a, Facultad de Ciencias Biolo´gicas,Pontificia Universidad Cato´lica de Chile, Santiago, ChileÃAuthor for correspondence: (email: firstname.lastname@example.org)
DNA methylation is one of the epigenetic and
environmental stimuli could produce effects, (ii) particular
hereditary mechanisms regulating genetic expression in
environmental agents as such stimuli, and (iii) that a genomic
mammalian cells. In this review, we propose how certain
persistent change be consequently produced in a population.
natural agents, through their dietary consumption, could
Depending on the persistence of the environmental stimuli
induce changes in physiological aspects in mammalian
and on whether the affected genes are imprinted genes,
mothers, leading to alterations in DNA methylation patterns
induced changes in DNA methylation patterns could become
of the developing fetus and to the emergence of new
persistent. Moreover, some fragments could be more
phenotypes and evolutionary change. Nevertheless, we
frequently methylated than others over several generations,
hypothesize that this process would require (i) certain key
leading to biased base change and evolutionary con-
periods in the ontogeny of the organism where the
We believe that a ﬁrst approach toward evaluating this
problem requires separating the phenomenon of the emer-
An old question in evolutionary biology is ‘‘how does var-
gence of an evolutionary novelty into two processes: (i) that
iation originate?’’ No matter how old this question is, the
responsible for the origin of a new character and (ii) that
controversy remains regarding whether (i) variability in pop-
maintaining such a character over generations (i.e., ﬁxation).
ulations appears exclusively by random mutations, a position
Such separation has been previously proposed by authors
defended by neo-Darwinism, or (ii) the formation of novel
such as Futuyma and Moreno (1988) and West-Eberhard
characters can, in some way, be induced by external environ-
mental forces. Current understanding of epigenetic modiﬁca-
As Darwin, most evolutionary biologists have concentrat-
tion of DNA shows that such controversy still exists. In this
ed almost exclusively on the second process, that is the form
sense, Jaenisch and Bird (2003) suggested that future lines of
in which an evolutionary novelty can be ﬁxed, not inquiring
investigation should place emphasis on the identiﬁcation of
into the problem of how evolutionary novelties originate.
the stimuli that can initiate evolutionary changes. They pro-
Variation among individuals and correlated differences in ﬁt-
posed that it is possible for external factors, such as dietary
ness became a central topic in Darwin’s theory (Endler 1986)
compounds, to lead to the accumulation of epigenetic changes
and thereafter, Neo-Darwinian theory interpreted changes in
over the years within populations. Given the recent evidences
allelic frequencies of populations instead of studying the or-
on mechanisms of epigenesis, here we propose that under
igin of new phenotypes (Nijhout et al. 1986).
certain conditions, such epigenetic changes could become
In accordance with the separation between origin and
persistent over generations and this could have evolutionary
ﬁxation of an evolutionary novelty, some authors state
that evolution is always a two-step process, ﬁrst involving
BLACKWELL PUBLISHING, INC.
developmentally mediated variation, and then selection,
At present, it is widely known that DNA methylation is
whose operation results in gene frequency changes (Wake
one of the epigenetic and hereditary aspects that regulate ge-
and Larson 1987; West-Eberhard 1998). In this sense, changes
netic expression in mammalian cells (Khosla et al. 2001).
arising because of alterations in early developmental process-
Furthermore, DNA methylation is capable of being modiﬁed
es, which, furthermore, could, in some cases, be environmen-
by the action of externally applied agents (Mac Phee 1998).
tally induced, can appear whether or not such changes could
Not all, but particular compounds found in nature could act
become ﬁxed and prosper in a population. Hence, in our
as such agents. Moreover, they could be capable of affecting
opinion, the diversity and evolution of species should be ex-
the evolution of organisms, inducing profound changes in
plained not only by those selective processes imposed by the
individuals and populations, perhaps with transgenerational
environment but also by the action of the environment as an
consequences. We hypothesize that, whereas certain condi-
inductor of genotypic and phenotypic variation, which is the
tions are required for this process to occur, it is a feasible
phenomenon. The task is to identify the conditions constrain-
Regarding the persistence of such epigenetic changes
through generations, long ago, Weismann (1893) stated that
Experimental evidences concerning alterations of methyl-
external inﬂuences may produce hereditary variations when
ation patterns, at least in mammals, are generally restricted
they are capable of modifying the determinants of the germ
to studies of the effects of synthetic compounds or dietary
plasm. Nevertheless, this could be only one of the ways
restrictions of food items containing the methyl group (see
through which environmental factors induce transgenerational
Laird and Jaenish 1996; Singal and Ginder 1999). Although
epigenetic changes. We recognize two ways for this to occur:
this is very important for understanding the mechanisms of
one is by dramatically modifying DNA aspects in the germ line
DNA methylation, from an evolutionary perspective, it is of
with transgenerational consequences, that is by means of pro-
greater relevance to ﬁnd compounds that are naturally in
ducing mutations or transgenerationally persistent epigenetic
contact with organisms; for example, those available for di-
modiﬁcations in the genome, and the other is through inducing
etary consumption, which, in addition, could produce alter-
ontogenetical variation at every generation, although not pro-
ations in patterns of DNA methylation in organisms.
ducing inheritance through the germ line. From our perspec-
In this article, focusing exclusively on the phenomenon of
tive, inductive environmental forces can act to create, through
how evolutionary novelties originate, we describe how in
one or both of these forms, new conformation of organisms,
mammals, certain natural agents could induce alterations
which also implies new possibilities within its surrounding
in particular mechanisms of regulation of gene expression in
environment. Jablonka and Lamb (1995) have named the
individuals, such as methylation patterns, and the further
range of the possible responses of individuals to new environ-
arising of new, specific phenotypes in subsequent generations,
mental challenges as the ‘‘reaction range’’ of individuals.
leading to evolutionary change. Nevertheless, we hypothesize
Based on his experiments in Drosophila, Waddington pro-
that this process would require (i) certain key periods in the
posed two new concepts related to the capacity of environ-
ontogeny of the organism where the environmental stimuli
mental inﬂuences to induce the appearance of new characters
could produce effects, (ii) particular environmental agents
in organisms and their maintenance over generations. First, in
as such stimuli, moreover, acting persistently, and (iii) that
the face of disturbing and external stressing inﬂuences, there
a persistent genomic change be consequently produced in a
are counteracting tendencies in development toward normal
adult conditions (i.e., canalization; Waddington 1959). Sec-
The ﬁrst requirement emerges because not all compounds
ond, whereas these counteracting tendencies exist, if a stress-
are capable of producing an effect on mothers that will have
ing stimulus is capable of developmentally modifying a strain
consequences on the fetus; the second emerges from the fact
of organisms, the derived population may evolve exhibiting
that an organism is not equally sensitive to outer stimuli
the modiﬁcation even in the absence of the stress (Wadding-
throughout ontogeny; and the third because transgenerational
ton 1952). He termed this process ‘‘genetic assimilation.’’
persistency of characters is ensured when it reaches the gen-
An important fact to notice is that, through these concepts,
omic level. Each of the three requirements presented will be
Waddington distinguished particular environmental stimuli
more extensively treated later in the text.
capable of inducing epigenetic changes, which are the ‘‘stress-ing’’ ones. McClintock (1984) also stated that a particularkind of stimuli producing stress lead to a genome’s reaction to
it, whose response may underlie formation of new species.
Furthermore, she stated that genome produces programmedresponses, although it is necessary to subject the genome
Experimentation on the problem of how evolutionary nov-
repeatedly to the same challenge in order to observe the na-
elties arise and the consequences on the genetic system
of exposition to an environmental stimulus have been the
focus of epigenetic studies in a variety of organisms, includ-
There are multiple isoforms of Dnmts, but all are encoded
ing Drosophila (Rutherford and Lindquist 1998), bacteria
by the same cytosine–Dnmt gene (Deng and Szyf 1998).
(Cairns et al. 1988), and yeast (Steele and Jinks-Robertson
Among these isoforms, Dnmt1o is a variant of Dnmt1 that
accumulates in oocyte nuclei during the follicular growth
Several types of epigenetic inheritance have been described
phase, and Dnmt3L is an isoform of Dnmt3a and Dnmt3b,
to date. Jablonka and Lamb (1995) have proposed three sys-
but that lacks Dnmt enzymatic activity and interacts with
tems of epigenetic inheritance: (i) steady-state systems, such as
Dnmt2a and Dnmt3b (Kierzenbaum 2002). Dnmt3L acts as a
Wright’s (1945) persistence of alternative cellular states as a
cofactor for de novo methylation of imprinted genes in the
result of changes in nuclear genes or in cytoplasmic constit-
female gametes and for the establishment of methylation im-
uents of the cell, (ii) structural inheritance systems, such as the
prints in oocytes (Hata et al. 2002).
maintenance through generations of the ciliary patterns in
It is worth noting that Dnmt1 is localized principally in
protozoa, albeit of the genetic constitution of the cells in-
somatic cell nuclei, but it is cytoplasmatic in the oocyte and in
volved (Nanney 1985), and (iii) chromatin-marking systems, or
the preimplantation embryo (Bestor 2000). However, the var-
those related to the transmission of specific patterns of the
iant Dnmt1o has transient nuclear localization in the eight-cell
chromatin structure (Holliday 1987; Jablonka et al. 1987).
stage, corresponding to the time when genomic imprints are
Specifically, the latter refers to non-DNA parts of the chro-
established (Howell et al. 2001). On the other hand, Dnmt3L
mosomes that are capable of binding proteins or additional
co-localizes with Dnmt3a and Dnmt3b in mammalian cell
chemical groups attached to DNA bases, which affect the
nature and stability of gene expression, now commonly
Given the crucial role of the diverse Dnmts in the epige-
named genomic imprinting. DNA methylation describes a
netic modiﬁcation of DNA, it is of great interest to know
postreplicative modiﬁcation, in which a methyl group is add-
whether there are environmental substances capable of mod-
ed to a DNA residue in a covalent manner (Laird and Jaenish
ifying the intracellular levels of such enzymes or their patterns
1996); for this reason, it is a form of genomic imprinting. The
of gene expression. Nevertheless, no studies have reported this
DNA methylation reaction is enzymatically catalyzed by
kind of interaction, which we suspect may have a role in
DNA methyltransferases (Dnmts) and takes place in 50 to 30-
relating environmental stimuli to DNA modiﬁcation. How-
oriented CG dinucleotides, which are known as CpG sites, at
ever, the recent ﬁnding that individual Dnmts can be tracked,
the carbon 5 of the cytosine ring (Singal and Ginder 1999).
and that their binding to genomic DNA can be quantiﬁed in
CpG islands are regions with a high frequency of CpG sites;
vivo in mammalian cells (Liu et al. 2003) can be enormously
these islands are often associated with genes, and are usually
helpful for determining the link between environmental com-
found in promoter zones (Gardiner-Garden and Frommer
pounds and the process of DNA methylation.
1987). CpG sites are not evenly distributed within the genome,and are preferentially unmethylated, regardless of the tran-scriptional activity of the associated gene (Bird 1986). As
other regions are normally methylated, patterns of genomic
DNA methylation can be distinguished along the genome
(Singal and Ginder 1999; Bestor 2000; Jones and Takai 2001).
Nevertheless, there is controversial information regarding
Roemer et al. (1997) were the ﬁrst to show reappearance in
whether methylation patterns are established because of
the progeny of modiﬁed characters in parents. In their exper-
the enzymatic activity of one or more Dnmts (Bestor 2000;
iments on rodents, the adult phenotype produced because of
the fusion of pronuclei with eggs of different genotypes was
There are at least three families of Dnmts described to
also observed in the oﬀspring. Furthermore, such transgene-
date: Dnmt1, Dnmt2, and Dnmt3. However, there is no
rational persistence of the modiﬁed characters was related to
agreement regarding whether each one plays a specific, dif-
altered methylation patterns that were, in turn, transmitted
ferential role in the process of DNA methylation (Bestor
through male gametogenesis. However, not all genes are
2000). It has been speculated that Dnmt3A and Dnmt3B are
equally capable of passing on changes in patterns of methyl-
responsible for the establishment of methylation patterns
ation. There is a particular class of genes, crucial for under-
during early development, whereas Dnmt1 is responsible for
standing the mechanisms of epigenetic inheritance, that are
the further maintenance of such patterns. Experiments con-
known to have relatively unchanged methylation patterns
ducted in vitro support this model, revealing that Dnmt1 has
over generations. These genes, named ‘‘imprinted genes’’, do
a preference for hemimethylated DNA as a substrate (Yoder
not seem to be affected by overall alterations in methylation
et al. 1997), whereas Dnmt3A and Dnmt3B act as a de novo
patterns that take place early in development (Constaˆncia et
methyltranferase, preferring unmethylated DNA (Yokochi
al. 1998). Such genes carry a molecular memory of their pa-
rental origin that is acquired early in the germ line (Surani
2001). This molecular memory is associated with specific me-
However, even if no one imprinted gene is affected when
thylation patterns in CpG islands of each allele, which con-
altered by an environmental signal, environmentally induced
sequently affect further genic expression (Costello and Plass
changes in methylation patterns could also become persistent
if such changes, and the environmental conditions allowing
Once the allelic differences in methylation of imprinted
the establishment of such changes, are both conserved
genes are deﬁned (during the establishment of germinal line
throughout generations. This could occur whenever there is
in the developing embryo), such differences generally remain
a concordance, an association between the environmental
stable in the somatic tissues (Constaˆncia et al. 1998). The
stimuli, the established DNA methylation patterns, and the
marking process of these genes appears to involve three stag-
resulting phenotype of an organism. For instance, if some
es: (i) the establishment of marks in gametes; (ii) the perma-
natural agent can induce the loss of methylation in genes and
nence of these marks during embryogenesis and in the adult
produce phenotypic alterations (e.g., those modiﬁcations
somatic tissues; and (iii) the erasure of marks in the early germ
emerging from the loss of methylation in Igf2), a standard
line (Razin and Cedar 1994). Conclusive information on the
phenotypic pattern will arise every time the specific environ-
way in which methylation in imprinted genes is initiated from
mental stimuli lead to the establishment of particular patterns
an unmethylated state during gametogenesis is still elusive
of methylation. Still, it is important to consider that this could
(Ferguson-Smith and Surani 2001). However, recent investi-
be a broader phenomenon, and environmentally induced
gations indicate that primordial germ cells are substantially
changes in methylation patterns could affect several other
methylated (which corresponds to the same pattern in somatic
imprinted genes as well. As a result, an environmental stim-
cells) before they colonize gonads and become demethylated
ulus would bias the phenotypic change toward certain types
around the time of entry into the gonads (Hajkova 2002). An
incomplete deletion of marks during gametogenesis would
Nevertheless, the consequences of altering DNA methyl-
explain the inheritance of the parental epigenotype (Reik et al.
ation toward specific persistent patterns could imply mutation
in those specific segments of the genome. For instance, itis known that a methylated cytosine is half-way to thesubstitution of a cytosine for a thymidine. The completion ofconversion requires only a hydrolytic deamination reaction
(Singal and Ginder 1999). Therefore, if some methylated sites
are frequently methylated over several generations, it is pos-sible that an eventual base change from cytosine to thymidine
Imprinted genes may be susceptible to undergoing changes in
will occur more frequently than any other substitution. In
methylation patterns during preimplantational development
fact, CpG sites are hotspots for transitions from cytosine to
(Khosla et al. 2001). As imprinted genes tend to conserve
thymidine, generated by a spontaneous deamination of 5-me-
methylation patterns from one generation to the next, chang-
thyl cytosine to thymidine (Coulondre et al. 1978). The result
ing methylation patterns in these genes could lead to the ap-
would be, as mentioned by West-Eberhard (2003), that
pearance of the derived alterations in the future generations.
‘‘evolved sensitivity to environmental inﬂuence during gene
Therefore, if external agents are capable of inducing partic-
expression could inﬂuence susceptibility to certain kinds of
ular changes in methylation patterns in these genes, such
structural change during evolution.’’
changes could ﬂourish transgenerationally. Moreover, thiscould take place in the absence of the stimuli that initiallychanged its methylation pattern, generating a process that
would be a kind of Waddington’s ‘‘genetic assimilation’’ but
Changes of methylation patterns in certain imprinted genes
The ﬁrst condition for our statement on environmentally in-
can generate associated specific phenotypes (see Morison et
duced evolution is that the process must occur early in on-
al. 2001 for examples). Particularly interesting, from our per-
togeny, before or during the establishment of the germ line in
spective, is the Beckwith–Wiedemann syndrome. Researchers
metazoa. This is important for two main reasons: ﬁrst, be-
suspect that this syndrome is related to the loss of imprinting
cause eventual reprogramming of methylation patterns in the
in Igf2, and is characterized by somatic overgrowth, macro-
germ line can be transmitted to the progeny (Surani 2001),
glossia, abdominal wall defects, visceromegaly, and an in-
and second, because during development, there is an en-
creased susceptibility to childhood tumors (Caspary et al.
hanced susceptibility of the organism to the action of outer
1999). Therefore, in this case, a change in methylation pat-
compounds, with greater consequences in the adult than when
terns in a single gene can lead to phenotypic changes in several
the same stimulus occurs later in ontogeny (Amzallag 2000).
With regard to the latter statement, Gould and Lewontin
(1979) have emphasized that during the early ontogenetic
that allow such organization to take place (Maturana-
stages of complex organisms, ‘‘differentiation of organ sys-
Romesı´n and Mpodozis 2000). An experimental approach
tems and their integration into a functioning body is such a
to such a statement comes from Clark and Galef (1995), who
delicate process so easily derailed by early errors, with accu-
proposed that daughters tend to resemble their mothers not
only because both share a relatively large proportion of their
The morphogenic process of an organism is basically the
genes but also because they tend to have similar histories of
product of a three-way interaction between the environment,
genetic factors, and those characteristics that emerge from
Applying this view to DNA methylation, reproduction
a self-organized dimension created by development itself
plays a key role in passing on those changes in patterns of
(Amzallag 2000). The establishment of methylation patterns
methylation that could eventually arise during early stages of
during early development (as well as other processes in
the ontogeny. Reproduction, in addition to conserving the
morphogenesis) also depends on the immediate environment
pattern of DNA methylation of an organism’s genome
experienced by the embryo. These methylation patterns will
throughout generations in a lineage, will also conserve the
guide the formation of particular cell types by controlling
conditions allowing such patterns of methylation to be es-
gene expression (Holliday 1998), therefore biasing further
tablished in every generation. Hence, for a mammal to be
formed from a zygote, and for development to take place
In mammals, patterns of methylation are established for
generating a phenotype similar to the parental phenotypic
the entire genome at least three times during development.
pattern, the process requires not only the genetic content that
The periods in which reprogramming of methylation patterns
provides a zygote with the potential to become an adult but
takes place are: (i) before the implantation of the embryo, (ii)
also a surrounding environment for the embryo, which en-
during the development of the germ line (Reik et al. 2001),
sures the occurrence of appropriate methylations, at key pe-
and (iii) during the period beginning soon after blastocyst
riods of time during the embryological process.
implantation (Constaˆncia et al. 1998) until gastrulation (Mac
Nevertheless, in mammals, despite the fact that the uterus
Phee 1998). Before blastocyst implantation, a great part of the
acts as a buﬀer for either mechanical or chemical perturbat-
DNA is demethylated (Dean et al. 1998); thus, the DNA of
ions on the developing embryo, making the developmental
blastocysts hardly shows methylation (Mac Phee 1998).
process more isolated from environmental perturbations than
Between blastocyst implantation and gastrulation, there is a
in other taxa, the development is still susceptible to particular
wave of de novo methylations that restore the overall me-
perturbations. Maternal effects such as variations in the
thylation patterns, which is retained in the somatic cells of
hormonal status of a mother are capable of affecting the
animal for the rest of its life (Mac Phee 1998). In the germ
microenvironment in which the fetus develops (Clark and
line, reprogramming takes place by overall demethylations
Galef 1998) and, consequently, its later ontogenetic processes
and methylations of the genome (Constaˆncia et al. 1998). In
(Bernardo 1996). For example, studies have shown that
mice, primordial germ cells undergo an overall demethylation
differential exposition to hormones can affect characters of
process in early development until day 13 or 14 (Reik et al.
the embryo. Clark et al. (1993) and Vandenbergh and Hug-
2001). Later, during gametogenesis, there is a de novo me-
gett (1994) demonstrated that the intrauterine position of
thylation event until the previously observed high levels of
female rodents affects the sex ratio of their litters, which is
methylation in the zygote (Mac Phee 1998), oocyte, and
because of differential prenatal exposure to steroidal hor-
sperm genomes (Reik et al. 2001) are reached. It is likely that
mones, which in turn depends on the gender of neighboring
both demethylations taking place during the ﬁrst stages of
postzygotic cleavage, and methylations occurring after im-
Besides, the hormonal state of the mammalian female can
plantation, are important in removing acquired epigenetic
be strongly inﬂuenced by the environment through com-
modiﬁcations, especially those acquired during gametogenesis
pounds that are naturally found in her diet (Nagao et al.
2001). In this sense, it has been reported that feed toxicants, ordietary imbalances of specific nutrients, can alter the compo-sition of oviductal and uterine secretions (McEvoy et al.
Thus, the establishment of methylation patterns in the
embryo is a process that depends directly on the environmentin which it takes place, that is the intrauterine environment,
Reproduction involves the conservation in the progeny not
but also indirectly on the surrounding environmental signa-
only of the structure required to carry out the self-conserved
ling, which, in some way, alters such an intrauterine environ-
organization represented by the organism but also the pres-
ment. Accordingly, perturbing the intrauterine environment
ervation of the structural characteristics of the environment
while early development takes place could bring about
consequences in the establishment of methylation patterns,
could also interpret this cell transformation as alterations in
with the corresponding phenotypic repercussions.
methylation patterns. Some evidence for this phenomenoncomes from studies in chicken liver, where estrogens appear toact in the regulation of expression of the vitellogenin I and II,
and VLDL II genes, through changes in patterns of methyl-
ation of estrogen-responsive element sites (Edinger et al.
1997). It has also been shown that neonatal exposure to DESand adult ovary hormones produces abnormalities in the de-
Our second condition is that only particular compounds in
methylation of the lactoferrine promoter, which shows that
nature could act as environmental inputs for environmentally
either hormonal xenobiotics or natural hormones are capable
induced evolution to take place. The early embryo is exqui-
of triggering impairments during the development of organs
sitely sensitive to alterations in its environment (McEvoy et al.
2001). Nevertheless, not every compound with which a mam-
It has been reported that environmental estrogens can also
malian mother has contact in nature is capable of altering the
produce direct effects on DNA methylation patterns. For ex-
embryo environment, although some compounds could lead
ample, administration of the phytoestrogens cumestrol and
to alterations in mammalian hormonal features. Furthermore,
equal to newborn mice can enhance methylation and produce
we believe that some environmental compounds can, in ad-
inactivation in the proto-oncogene H-ras (Lyn-Cook et al.
dition to altering the hormonal status of a mammalian moth-
1995). In addition, Day et al. (2002) demonstrated that me-
er, be in turn capable of affecting important processes during
thylation patterns can be altered in 8-week-old mice that
the early development, including the establishment of me-
consumed high quantities of genistein.
thylation patterns in the embryo. Among those environmen-
With respect to hormonal effects early in development,
tally available compounds capable of affecting the hormonal
Holliday (1998) was the ﬁrst to envisage a possible link be-
status of a mammalian mother, there are some of synthetic
tween hormone action and establishment of DNA methylat-
origin, or xenobiotics (Danzo 1998) and of natural origin,
ion in mammalian embryos. He proposed that the effect of
such as phytoestrogens. The latter refers to secondary met-
teratogens on a mother might disrupt the normal distribution
abolites produced by plants (Croteau et al. 2000; Yu et al.
of DNA methylation in a developing fetus, producing devel-
2000) that produce estrogenic action at a variety of levels in
opmental abnormalities or defects that can appear in the
animals (McLachlan 2001). Phytoestrogens are readily avail-
subsequent generations. Newbold et al. (2000) reported that
able in the environment for animal consumption and their
after administering DES to pregnant rats during early post-
physiological, hormonal, and nonhormonal effects in animals
implantational development and neonatality, a greater sus-
have been studied to some extent (Levy et al. 1995; Santell
ceptibility for specific tumor formation in rete testis and
et al. 1997; Boettger-Tong et al. 1998; Milligan et al. 1998;
reproductive tract tissues occurred in F1 and appeared further
Gallo et al. 1999). Some phytoestrogens such as genistein and
in the non-DES exposed F2. These authors speculated that
daidzein belong to a class of ﬂavonoids, the so-called isoﬂav-
this transgenerational phenomenon could implicate epigenetic
ones (Liggins et al. 2000). The consumption of isoﬂavones can
alterations that were transmitted through germ line, including
elicit uterotrophic and mammatrophic effects in mice and on
changes in methylation patterns. Although this ﬁnding strong-
the hypothalamic/pituitary axis as well (Santell et al. 1997). In
ly suggests alteration and further transmission of a genomic
humans, it has been reported that the consumption of phyt-
change through germ line across more than one generation in
oestrogens affects levels of the sex hormone-binding globulin,
response to an early exposition to an estrogenic compound,
which regulates the bioavailability of steroidal sex hormones
there is still missing evidence on the mechanism behind this
process and whether it implies changes in DNA methylation
Changing the hormonal status in mammals could have
consequences beyond the physiologic level. McLachlan (2001)
In the experiments of Newbold et al. (2000), the trans-
suggested that estrogens could play a role in programming or
generational persistence of the enhanced susceptibility to
imprinting those genes involved in cell proliferation, diﬀeren-
tumor formation takes place when mothers are exposed to
tiation, or survival, either directly or through related signaling
DES after embryo implantation; however, estrogens play an
pathways. He also proposed that an estrogenic chemical may
important role even before implantation occurs. The implan-
directly imprint a gene through a process leading to persistent
tation process involves complex interactions between the
genetic change, probably at the level of DNA methylation. In
blastocyst and the uterus (Paria et al. 1993). Uterine preim-
this sense, Barrett et al. (1981) suggested that diethylstilbestrol
plantational estrogen secretions are essential for activating the
(DES), a powerful estrogenic synthetic compound, could
blastocyst of Mus musculus for further implantation, which
transform cells by mechanisms other than punctual muta-
is not possible if estrogen secretions are prevented by
tions, frameshift mutations, or small deletions. Currently, one
ovariectomization (Paria et al. 1998). Nevertheless, just as
estrogenic stimuli are needed for normal development, pre-
cysts, which could occur through membrane-mediated estro-
implantational exposure to synthetic estrogenic compounds
gen actions, directly induced by isoﬂavones in uterine
can lead to phenotypic alterations. For instance, Takai et al.
secretions, or mediated by other compounds secreted in the
(2000) reported that in utero preimplantational exposure of
uterine epithelia such as 4-OH-17b-estradiol. The formation
rodent embryos to the synthetic estrogen bisphenol-A leads to
of this compound in uterine epithelia could be related to
an increased body mass of the animals at weaning. Further-
plasmatic isoﬂavone content, although no studies have at-
more, Wu et al. (2004) have recently shown that in vitro early
tempted to detect such compounds in uterine secretions, or
exposure to the environmental contaminant 2,3,7,8-tetra-
showed that its high consumption can alter the production of
chlorodibenzo-p-dioxin can indeed alter DNA methylation
cathecolestrogens in the uterine epithelia.
patterns in preimplantational embryos. Interestingly, those
Although there is strong evidence suggesting that the hor-
genes changed, H19 and IGF-2, were imprinted genes.
monal status of mammalian mothers can be an important
Although it is not known whether compounds with est-
feature related to the establishment of methylation patterns in
rogenic action (CEA) inside the uterus could act directly upon
early embryos, so far, there is no concluding evidence of this.
the developing embryo, or via intermediaries, it is possible
We believe that an investigation on this subject should be
that the relationship between estrogenic stimuli and methyl-
performed in order to uncover the aspects behind an eventual
ation in the preimplantational embryo is mediated by the ex-
epigenetic role of estrogenic compounds (both animal pro-
pression of c-fos. While on the one hand it is known that c-fos
duced, plant produced, and synthetic) on developmental
directly regulates the dnmt1 transcription, increasing Dnmt1
processes, in particular, on the establishment of methylation
levels (Bakin and Curran 1999), on the other, the induction of
c-fos is a response attributed to membrane-mediated estrogenactions (Das et al. 2000). Through this mechanism, whichprovides an alternative pathway to the classical estrogen re-
THE ‘‘GENOMIC CHANGE’’ REQUIREMENT FOR A
ceptors a and b, CEA could trigger responses, as has been
observed in pancreatic b cells (Nadal et al. 2000). In summary,the membrane-mediated estrogenic actions would ﬁrst induce
The third requirement that we propose for the environmental
c-fos and then trigger the activation of the Dnmt1 enzyme.
and hormonal induction to become an evolutionary process is
Furthermore, in blastocysts, this indirect and membrane-
that genomic change should be achieved. Evolutionary
mediated relationship between estrogenic stimuli and c-fos
change in the morphogenetic process must arise from chang-
activation could also occur. In preimplantational blastocysts,
es in patterns of regulation and interaction during ontogeny
Paria et al. (1998) demonstrated that latent blastocysts can be
(see discussion by Atchley 1987). Such a connection gains
activated if they are incubated in vitro with 4-OH-17b-estra-
special importance when considering that the patterns of reg-
diol, a catecholestrogen synthesized from 17b-estradiol in
ulation and interaction occurring at early stages in ontogeny
uterine luminal epithelia by the action of the hydrogen-2/hid-
could, even in mammals, be susceptible to environmental
roxilase-4 enzyme. This response to 4-OH-17b-estradiol could
also occur via a pathway distinct from the classical nuclear
Nevertheless, the question arising at this point goes beyond
estrogen receptors (Paria et al. 1998). In addition, Paria et al.
the relationship between the environmental stimuli and even-
(1998) found that 4-OH-17b-estradiol increases with the ep-
tual epigenetic consequences on DNA methylation. The chal-
ithelial growth factor (EGF) receptor. Interestingly, other
lenge is to know how an eventual change in DNA
studies have demonstrated that an increase in the EGF re-
methylation patterns could become persistent and evolution-
ceptor may also be related to activation of c-fos (Kamiya et al.
ary. Besides, another question arises, regarding the definition
1996). On the other hand, a direct induction of c-fos by est-
of evolutionary change. Is persistence in the conditions al-
rogen has also been shown in different cell types (Allen et al.
lowing the establishment of changed methylation patterns
1997; Garcia et al. 2000), which occurs via an estrogen re-
across lineages a sufﬁcient attribute for such changes to be
ceptor element present in this gene (Hyder et al. 1992). Thus,
considered as evolutionary, or do such changes need to reach
estrogenic stimuli could induce c-fos, either directly, through
the threshold of mutation at the genomic level?
a gene receptor, or indirectly through membrane-mediated
It is true that genomic mutational change ensures a great
degree of persistence through generations. However, persist-
Furthermore, phytoestrogens could also induce c-fos and
ence can also be the result of two processes, as previously
consequently alter methylation patterns in cells. A study sup-
mentioned: (i) the environment could persistently trigger,
porting this view demonstrated that the intake of genistein in
generation after generation, a specific change in methylation
ovariectomized female rodents induced the expression of the
patterns, or (ii) persistence could be present in intrinsic fea-
RNA messenger of c-fos in the uterus (Santell et al. 1997).
tures of the organisms as, for instance, the stable nature of
Hence, we suspect that phytoestrogens can also act on blasto-
Given the special feature of imprinted genes regarding
In this sense, if a natural population of rodents is suddenly
possessing methylation patterns that are more stable across
subjected to a high intake of phytoestrogens, it is feasible
generations than other genes, persistence could be achieved
to hypothesize that such a high intake by pregnant rodents
through changing methylation patterns of imprinted genes. In
could inﬂuence the normal reproductive process, altering
this view, such changes in imprinted genes could have the same
the mother’s hormonal status, the intrauterine signaling,
evolutionary value of mutations, given that there is an asso-
and, consequently, the establishment of DNA methylation
ciated character variation with the changes, and because of the
patterns in embryos. The resulting phenotypes will be in ac-
persistence of these changes throughout generations. Thus, the
cordance with the particular pattern of DNA methylation
definition of ‘‘evolutionary change’’ at this point becomes
achieved as a consequence of the environmental stimuli,
blurred. What is true is that persistence through generations
represented in this case by phytoestrogens. As a result, such
could be achieved in alternative ways to genomic mutation.
changes in methylation patterns will persist in the population
Nevertheless, speaking in terms of genomic mutation, this
if the organisms are constantly subjected to this same
could be achieved when the persistent change in methylated
environmental input and consequently, the achieved pheno-
cytosines bias to specific mutations, as previously mentioned.
types will also persist throughout generations. Nevertheless, it
Regarding the frequency of eventual mutations derived
is important to point out that such a newly formed phenotype
from changes in methylation patterns, given that such
must not be considered to be associated with any adaptive
changes can be environmentally induced, they cannot be con-
goal; on the contrary, the new forms of organisms will ﬁt
sidered to be at random. Therefore, we can expect that in these
within the environment they live, resulting from an environ-
cases, the appearance of mutation will be in greater frequency
mental input that leads to standardized phenotypes in
than when mutation is considered to be at random. In fact,
concordance with the environmental stimuli that produced
there is a 12-fold higher than normal mutation rate for the
them. In the particular case of imprinted genes, changing
conversion of the methylated form of CpG to TpG and CpA,
methylation patterns on those genes could imply transgene-
which reduces the occurrence of CpG to about 20% of its
rational persistence of epigenetic changes in the absence of
expected frequency in vertebrate genomes (Sved and Bird 1990).
the environmental input that initially produced them.
Despite the evidence suggesting that the environment,
Because the early stages of ontogeny play key roles in the
through the action of naturally consumed agents, can alter
establishment of phenotypic variation, it is important to
the developmental process to the point that the emerging
determine how environmental signals (particularly CEA) are
alterations can be inherited as evolutionary change, conclusive
involved in the developmental process. Nevertheless, a com-
information is still elusive. Evidence in the direction of
plete understanding of this involvement is difﬁcult at this
genomic change derived from alterations in methylation
time. One of the complications is that the mechanisms
patterns is needed for our hypothesis to be plausible in the
through which estrogen and CEA bring about physiological
classic view of the meaning of evolutionary change.
actions are not yet clearly understood (Nilsson et al. 2001).
Further studies on the effects of CEA on organisms, especiallyduring early stages of ontogeny, are needed to provide new
insights, and to help in the understanding of the impact of this
class of compounds on ecosystems in general (McLachlan
2001), and, particularly, on the physiologically relevant evo-lutionary processes that guide the formation of organisms and
Holliday (1998) proposed that teratogens could target mech-
anisms that control patterns of DNA methylation in partic-ular regions of the genome of developing embryos, modifying
methylation patterns of the same DNA sequence in somatic
The authors wish to thank Roxanna Gomez, Paula Neill, and Renee
cells, leading to a developmental alteration, and subsequently
Hill for their help in the preparation of the manuscript and linguisticrevision. We also thank Dr. Gloria Calaf for her critical review of the
producing changes in germ line cells. Moreover, if such
manuscript and valuable suggestions. Carlos Guerrero-Bosagna
altered methylation patterns are eventually transmitted to a
gratefully acknowledges CONICYT and also INTA and Nestle´
subsequent generation, the same type of defect might be seen
(Holliday 1998). Phytoestrogens could act in the same man-ner, but with the peculiarity that they are naturally available
for consumption by many organisms. Phytoestrogens arepresent in high quantities in food items commonly included in
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