Alcohol & Alcoholism Vol. 32, No. 2, pp. 113-128, 1997
INVITED REVIEW
THE TERATOGENIC EFFECTS OF ALCOHOL FOLLOWING EXPOSURE
DURING PREGNANCY, AND ITS INFLUENCE ON THE CHROMOSOME
CONSTITUTION OF THE PRE-OVULATORY EGG
M. H. KAUFMAN
Department of Anatomy, University Medical School, Teviot Place, Edinburgh EH8 9AG, UK
(Received 19 August 1996; in revised form 21 October 1996)
Abstract — Much information has emerged over the years concerning the teratogenicity of acute and
chronic alcohol exposure during pregnancy. Both alcohol and its primary metabolite, acetaldehyde, are
teratogenic. Exposure during pregnancy may lead to fetal alcohol syndrome (FAS), and this is said to
occur in a substantial proportion of infants born to mothers who are chronic, heavy daily drinkers. Such
infants usually survive to birth but arc mentally retarded, often display growth retardation and
additionally display a characteristic range of clinical features, principally craniofacial abnormalities and
neurological damage. We have recently been interested in the effect of exposure of pregnant female mice
to a single high level of alcohol during pregnancy, equivalent to an episode of 'binge' drinking, on the
optic nerve, and believe that our findings, which are outlined in the first part of this review, may shed
important light on the pathogenesis of some of the ocular features characteristically seen in infants with
this syndrome.
What is not generally appreciated, is that exposure to alcohol and other 'spindle-active' substances
that have a similar action on the meiotic spindle apparatus during the menstrual cycle before conception
can induce chromosome segregation errors in the ovulated oocyte. The successful fertilization of such
eggs consequently results in the production of aneuploid embryos, which have a very high chance of
being spontaneously aborted during the first trimester of pregnancy. Those relatively few aneuploid
conceptuses that survive to term invariably show moderate to severe degrees of mental retardation,
craniofacial and other abnormalities, as well as having a significantly reduced life expectancy. The
findings from our experimental studies that have been carried out in mice draw attention to important
principles which are of general applicability to the situation in the human. These findings, and our
conclusions drawn from them, arc discussed in detail in the second part of this review.
1. GENERAL OBSERVATIONS ON THE
TERATOGENIC EFFECT OF EXPOSURE TO
ALCOHOL DURING EARLY PREGNANCY
of delivering an infant with an alcohol-related
mental and psychomotor deficit than matched
controls. At higher levels of intake (>59g/day)
the risk of long-term sequelae was substantially
The teratogenicity of ethyl alcohol (alcohol, increased. In some studies, the average daily
ethanol) has been known since antiquity (Warner consumption of alcohol during pregnancy was
and Rosett, 1975; Abel, 1984; Plant, 1985), though considerably higher (e.g. 140 ± 72 g, mean ± SD,
only over the last 20 years or so has it been fully see Spohr and Steinhausen, 1984). While the data
appreciated that infants born to mothers with a in the latter study were not clearly interpretable in
high alcohol intake during pregnancy run the risk terms of a dose-response relationship, a consistent
of exhibiting a characteristic pattern of craniofa- decrease in performance in a range of psychocial features which are often associated with motor tests was noted with increasing alcohol
growth retardation. According to Streissguth el exposure.
al. (1984), those individuals with an intake of
The greatest cause for concern, however, relates
>30g/day of absolute alcohol were at greater risk to the substantial incidence of brain dysfunction
113
1997 Medical Council on Alcoholism
114
M. H. KAUFMAN
seen in these infants (Palmer et al., 1974; Barry
and O'Nuallain, 1975; Smith et al., 1976; Clarren
et al., 1978; Streissguth et al., 1978; Wisniewski et
al., 1983; Pratt, 1984), where narrowing of the
head (Tenbrinck and Buchin, 1975), abnormal
EEG (Havlicek and Childaeva, 1976) and cortical
atrophy (Jones et al., 1974) have all been reported.
They also commonly display evidence of pre- and
postnatal growth retardation (Kyllerman et al.,
1983; Streissguth et al., 1984; Abel and Hannigan,
1995) and are usually moderately mentally
retarded with an average IQ of about 65-70,
depending on how the sample is collated (see
Streissguth et al., 1984). In other studies (Spohr
and Steinhausen, 1984), a wider range of IQ scores
were reported. In severe cases, this condition can
result in the birth of a stillborn infant or the death
of the infant during the neonatal period.
This relationship between alcohol consumption
during pregnancy and the birth of mentally
retarded infants often with an abnormal facial
appearance was first highlighted in the late 1960s
and early 1970s (Lemoine et al., 1968; Jones and
Smith, 1973; Jones et al., 1973) and termed the
fetal alcohol syndrome (FAS) (for additional early
references, see Hanson et al., 1976; Mulvihill and
Yeager, 1976; Clarren and Smith, 1978; Clarren,
1981; Spohr and Steinhausen, 1984; for bibliography of alcohol and reproduction, and fetal
alcohol exposure and effects, see Abel, 1982,
1984, 1990; for recent review of the literature, see
Abel, 1995). These infants usually have characteristic cTaniofacial features such as an abnormally
long philtrum, a short stubby nose and short
palpebral fissures associated with a positive
history of maternal alcoholism (for incidence and
range of clinical features, see Clarren and Smith,
1978; Spohr and Steinhausen, 1984). Abnormal
dermatoglyphic configurations have also been
reported in FAS (Qazi et al., 1980).
In an early study, Jones et al. (1974) stated that
FAS occurs in 30—45% of infants born to chronic,
heavy daily drinkers. A more recent study
indicates that the incidence is probably closer to
4.3—4.7% among 'heavy' drinkers, depending on
how the figures are analysed (Abel, 1995). While
the criteria for diagnosing this condition are now
more precise than formerly, FAS still represents a
substantial cause of perinatal morbidity. The
worldwide incidence of this condition is said to
be about 0.97 per 1000 live births in the general
obstetric population (Abel, 1995), and a similar
incidence has been reported in the west coast of
Scotland (Beaftie et al., 1983).
A recent review of the US and European
literature suggested that 'the major factor associated with FAS is low socio-economic status,
rather than racial background. Low socio-economic status and heavy alcohol consumption are
both associated with smoking, poor nutrition, poor
health, increased stress and use of other drugs, and
it is likely that they exacerbate the effects of heavy
alcohol intake' (Abel, 1995). This study further
emphasizes that, because criteria for determining
'heavy drinking' vary to such a degree between
studies, any attempt to make comparisons between
them—for example, on the incidence of FAS—is
likely to be extremely tentative (for a comprehensive set of guidelines regarding the definition of
'alcoholism', see Criteria Committee, National
Council on Alcoholism, 1972).
It is unclear at the present time whether alcohol
has a single mode of action at the cellular or
subcellular level, or the underlying mechanism
may vary according to the organ system under
examination. Our attempts to analyse the influence
of prenatal exposure to ethanol on one component
of the visual system using an animal model (see
below) represents an example of the approach
being taken to investigate this complex problem. It
is hoped that this may eventually provide insight
into the pathogenesis of the ocular neurological
damage sustained in a high proportion of infants
with FAS.
a. Experimental studies carried out to investigate
the teratogenicity of ethanol
Numerous experimental studies have been
carried out over the years using a wide range of
animal species to investigate the underlying
mechanism of action of ethanol. This agent has,
for example, been given to experimental animals
by intraperitoneal and intravenous injections, by
gavage, in the drinking water or in the diet
(Papara-Nicholson and Telford, 1957; Sandor and
Amels, 1971; Tze and Lee, 1975; Kronick, 1976;
Rosman and Malone, 1976; Chernoff, 1977;
Henderson and Shenker, 1977), and all have
produced some degree of developmental and/or
growth retardation as well as an increased incidence of embryonic losses and perinatal mortality.
During pregnancy, ethanol is metabolized in the
TERATOGENIC EFFECTS OF ETHANOL
maternal liver to acetaldehyde, mainly by the
cytosolic alcohol dehydrogenase, while acetaldehyde is further oxidized to acetic acid by aldehyde
dehydrogenase, both substances freely crossing
the placenta to enter the fetal circulation (Dilts,
1970; Kesaniemi and Sippel, 1975; Sippel and
Kesaniemi, 1975; Kaufman and Woollam, 1981;
Guerri et al., 1984). Ethanol is cleared more
slowly, however, from the fetal than the maternal
circulatory system (Cook et al., 1975). Indeed, its
rate of elimination from the fetal circulation is less
than half that observed in the mother (Wagner et
al., 1970), principally because the activity of
alcohol dehydrogenase in the fetus is far less than
that of the adult; alcohol dehydrogenase activity is
first detectable in the rat fetus on about the 18th
day of gestation, after which time it increases to
~ 2 5 % of the adult activity at birth (Raiha et al.,
1967; see also Sanchis and Guerri, 1986; Boleda et
al., 1992); aldehyde dehydrogenase activity is
absent on the 15th day of gestation in the fetal rat
liver, but a low level has been noted at term
(Sanchis and Guerri, 1986).
The teratogenic effect of exposure to alcohol in
experimental animal studies appears to be both
species- and strain-dependent (Schwetz et al.,
1978; Schenker et al., 1990; Zajaz and Abel,
1992), with rodents being used in the majority of
these studies (Kronick, 1976; Chernoff, 1977,
1980; Brown et al., 1979; Randall and Tabakoff,
1979; Abel et al., 1981; Sulik et al., 1981; Randall
and Riley, 1981; Sulik and Johnson, 1983; Sulik,
1984; Cook et al., 1987; Gilliam et al., 1990;
Garro et al., 1991; Middaugh and Boggan, 1991;
Kotch et al., 1992; Pinazo-Duran et al, 1993).
Similar teratogenic effects have also been reported
following the exposure of pregnant mice to
acetaldehyde, the primary breakdown product of
ethanol (O'Shea and Kaufman, 1979, 1981;
Webster et al., 1983). This finding should not be
surprising considering that ethanol is known to be
rapidly metabolized by the adult (Akabane, 1970;
Truitt and Walsh, 1971; Rahwan, 1974), and its
breakdown products indicated as primarily responsible for the teratogenic effects observed (Hanson
et al., 1977; Campbell and Fantel, 1983). In the
case of ethanol and acetaldehyde, a marked doserelated effect is invariably observed. It is relevant
to note that abnormalities of the central nervous
system have been particularly commonly encountered following exposure to alcohol (Hammer and
115
Schiebel, 1981; Goodlett et al., 1989; Miller,
1993).
In the most recent studies, exposure to alcohol
at regular intervals during pregnancy in a nonhuman primate (Macaca nemistrina) revealed a
dose-related neuroteratogenic deficit in the number of cerebellar Purkinje cells (Bonthius et al.,
1996), being consistent with the generalized and
permanent alcohol-induced reduction in neuronal
number reported by others. Such an effect had
previously been reported in the hippocampus
(Barnes and Walker, 1980), somatosensory cortex
(Miller and Potempa, 1990), cerebellum (Hamre
and West, 1993), retina (Clarren et al., 1990) and
olfactory bulb (Bonthius et al., 1992). Neuronal
loss has also been induced in areas known to
influence specific behaviour patterns, such as
inducing spatial memory deficits due to loss of
CA1 pyramidal cells in the hippocampus (Goodlett et al., 1992).
A considerable literature is also now accumulating regarding the effect of exposure of cells in
tissue culture to ethanol. For example, cortical
neurons and glial cells isolated from fetal and
newborn rat brains from pregnancies where the
mother has been exposed to ethanol either before
or during pregnancy have been incubated for
various periods of time in the presence of this
agent. Delayed nerve cell maturation, assessed
using a variety of parameters, was reported (Ledig
et al., 1991; see also Renau-Piqueras et al., 1989;
Guerri et al., 1989). Neural cells maintained in
tissue culture have also been used to explore the
cellular and molecular mechanisms which underlie
ethanol intoxication, tolerance and withdrawal
(Charness et al., 1984). Additional studies have
noted that exposure of a variety of other (nonneural) cell types to ethanol in tissue culture can
not only interfere with chromosome segregation,
but also induce chromosome damage (Obe and
Ristow, 1979; Obe et al., 1977, 1986; see also
section 2, below).
Culture of rat embryos for 4 h in serum isolated
from individuals shortly after consuming alcohol
(giving a serum ethanol level of 115 mg/dl),
followed by 44 h in alcohol-free serum, compared
to controls maintained for 48 h in normal alcoholfree serum, revealed a teratogenic growth-retarding effect of exposure to this agent. A similar
effect was observed when rat embryos were
incubated in culture medium containing a similar
116
M. H. KAUFMAN
level of ethanol. Toxic effects of acetaldehyde
were only seen at concentrations of 20 ug/ml, in
the absence of alcohol (Beck et al., 1984).
b. Influence of prenatal exposure to ethanol on
the optic nerve
Because the clinical and experimental literature
in relation to the teratogenicity of ethanol is now
so well documented (see above), it seemed
appropriate here to review only our most recent
experimental findings on the effects of prenatal
exposure to ethanol on the optic nerve of the
mouse, as we believe that this is a model which
allows us to study the pathogenesis of the ocular
neurological damage characteristically seen in
FAS.
It is important to note that the optic nerve is not
a true peripheral nerve, but a central tract whose
fibres differentiate mainly from the neural retina,
which is itself a derivative of the embryonic
forebrain (Hamilton and Mossman, 1972). It is
formed by neurons of the ganglion cells of the
retina as they converge at the optic disc to leave
the eyeball near its posterior pole. From here, the
nerve runs to the anterolateral horn of the optic
chiasma. After the nerve leaves the eyeball, it
becomes ensheathed by extensions of the meningeal coverings of the central nervous system. This
relationship extends almost throughout the entire
length of the nerve with the exception of a small
portion at the chiasmatic end where both the dura
and arachnoid mater layers are deficient. The
relationship between the meningeal coverings of
the optic nerve allows the subarachnoid and
subdural spaces of the brain to extend around the
nerve; consequently any raised intracranial pressure affecting the brain also affects the optic nerve
(Williams et al., 1989). It is also the most
accessible of the central tracts, and therefore
ideal for experimental studies.
The optic nerve has been extensively studied to
determine the effects of ethanol on this system
(Cohen, 1967; Kennedy and Elliott, 1986; Chan et
al., 1991; Pinazo-Duran et al., 1993). Although
some morphometric studies have been carried out
on the long-term effect of acute prenatal exposure
of the optic nerve to ethanol in the mouse (Parson
et al., 1993, 1995; Ashwell and Zhang, 1994), no
systematic attempt had been made to investigate
its effect during the early postnatal period.
L Methodology employed. Morphometric studies were undertaken using optic nerves isolated
from various inbred strains of immature, juvenile
and adult mice. The cross-sectional areas of intact
optic nerves excluding their meningeal coverings
were determined, and a systematic random
sampling procedure (Mayhew, 1990) allowed the
numbers and diameters of the myelinated nerve
fibres present to be established. The distribution of
diameters of all the myelinated nerve fibres
studied in each optic nerve was then plotted, and
an estimate of the total myelinated nerve fibre
population in each optic nerve and their density
could then be calculated using a ratio technique
(Matheson, 1970; Mayhew, 1988; 1990)
ii. Findings from morphometric studies. Of the
variables analysed, no significant differences were
observed in the cross-sectional areas, total number
of nerve fibres present, and nerve fibre density
between the left and right optic nerves and
between any of the variables analysed between
the male and female mice (Dangata et al., 1995).
In the rodents that have been studied, the total
number of myelinated nerve fibres present in
various strains of mice has varied between 66 000
and 94 000 (Gyllensten and Malmfors, 1963;
Gyllensten et al., 1966; Dangata et al., 1995)
and 117 000 and 120 000 in the rat (Forrester and
Peters, 1967; Hughes, 1977), while the maximum
fibre diameter in the mouse was just over 1.9 urn
(Dangata et al., 1995). The small fibre size
diameter seen in rodents compared to that seen
in primates, for example, may reflect the fact that
rodents are more dependent on senses other than
sight for most of their activities.
Hi. Influence of alcohol on timing of onset and
progression of myelinogenesis. No myelinated
nerve fibres were observed in the mouse before
the fifth day of postnatal life. Once myelination
was initiated, however, it progressed very rapidly
during the early stage of postnatal development,
and slowed thereafter. The peak level of myelination corresponded with the age when the maximum number of myelinated nerve fibres was
measured, and occurred at the 16th week of
postnatal life (Dangata et al., 1996). It should be
noted that in the mouse the eyelids open ~ 12 days
after birth (Findlater et al., 1993), by contrast to
the situation in the human where this event occurs
during the seventh month of fetal life (Hamilton
and Mossman, 1972).
TERATOGENIC EFFECTS OF ETHANOL
Studies on the timing of onset and progression
of myelinogenesis (Dangata and Kaufman, 1997;
see also Tennekoon et al., 1977) have also proved
to be instructive, in that they have highlighted a
particularly subtle effect of prenatal exposure to
ethanol, namely that it not only causes a delay in
the onset of this process, but also interferes with
its normal progression. The spectrum of size
distribution of the myelinated nerve fibres was
broader in the matched controls that received
saline alone compared to the corresponding
ethanol-exposed group. With increasing age, the
population of small and medium-sized fibres was
greater in the experimental group than in the
controls, while in the case of the large diameter
fibres the reverse was observed (Y. Y. Dangata
and M. H. Kaufman, unpublished work).
iv. Possible mode of action of prenatal exposure
to alcohol on the optic nerve. The delay in the
onset and rate of progression of myelinogenesis in
the mouse, together with the reduction in mean
myelinated nerve fibre count and density observed
suggests that ethanol probably exerts its teratogenic effect either on the glial precursors of
oligodendrocytes or on their cytodifferentiation,
and that this could result in both a reduction in
their number and/or their competence to synthesize myelin. A similar effect was observed in the
rat optic nerve following combined pre- and
postnatal exposure to ethanol (Phillips, 1989;
Phillips et al., 1991; Phillips and Krueger, 1992;
see also Samorajski et al., 1986). It was speculated
that such an effect might explain the neurological
signs and severe visual dysfunction commonly
seen in infants diagnosed as having FAS (Phillips
et al., 1991; Phillips and Krueger, 1992). A
decrease in brain myelin and a delay in brain
myelin synthesis have also been reported following exposure of rats to acute doses of ethanol
(Lancaster et al., 1982, 1984).
When an attempt was made to determine the
long-term effect in the mouse of an acute prenatal
exposure to ethanol, no significant difference was
observed in the total myelinated axon population
before 9 postnatal weeks. However, between 9 and
15 weeks, the alcohol-treated group showed a loss
of ~ 2 5 % of myelinated axons, so that by 15
weeks the alcohol-treated group had significantly
fewer myelinated axons; axons of all sizes were
lost in the experimental group during this period.
This was also reflected in a trend towards a
117
smaller cross-sectional area of the optic nerve (see
Parson et al., 1995).
c. Comparison between clinical and experimental
findings
Despite obvious differences in total myelinated
axon count, and fibre diameter spectrum between
the mouse and the human optic nerve, it is clear
that the mouse optic nerve is particularly sensitive
to exposure to ethanol when this occurs during
mid-gestation. In this way, it appears to resemble
closely the sensitivity of the human fetal optic
nerve to this agent, since optic nerve hypoplasia is
one of the most characteristic features of FAS, and
is believed to contribute to the poor visual acuity
of infants with this condition. Up to 90% of
affected children have eye defects, and over half
of these have either unilateral or bilateral optic
disc hypoplasia, with the optic nerve-head of
affected children being significantly smaller than
that of normal children (Stromland, 1987; Chan et
al., 1991). It should also be emphasized that in
some instances hypoplasia of the optic nerve-head
may be the only visible sign of alcohol damage to
the central nervous system (Pettigrew, 1986).
While it has been established that the decrease in
area of the optic nerve-head is related to a loss of
axons in the nerve, and that this is directly related
to a concomitant loss of retinal ganglion cells
(Lambert et al., 1987), no similar postmortem
studies have been carried out on FAS children to
enable analysis of retinal ganglion cells or axon
counts in the optic nerve.
Using the same experimental model, which
attempts to compare the effect of an acute prenatal
exposure to ethanol with that described previously, Parson and Sojitra (1995) investigated
whether the axon loss previously reported in the
optic nerve was stable and specific to the central
nervous system. Optic, tibial and saphenous
nerves were isolated from 12- and 23-week-old
experimental and matched control mice in order to
determine their cross-sectional area, total number
of myelinated axons present and axon diameter
distribution. The studies confirmed previous findings regarding axonal loss in the experimental
group, but noted that no further loss occurred
between 15 and 23 weeks. No significant differences were observed, however, in any of the
parameters measured in the tibial and saphenous
nerves between the experimental and control
118
M. H. KAUFMAN
groups. The teratogenic effect of ethanol therefore
appears to be specific to the central nervous
system, with no obvious effect on the peripheral
nervous system.
No evidence of a peripheral neuropathy was
seen in this 'binge' model, nor have there been
instances of peripheral neuropathy reported in
cases of FAS, though in one model for this
condition subtle differences in the sciatic nerve of
rats fed alcohol throughout pregnancy and during
the first postnatal week were reported (Baruah and
Kinder, 1989). This is in marked contrast to the
situation observed in chronic alcoholic subjects,
where peripheral neuropathy is one of the
commonest clinical manifestations (Victor, 1975;
Junrunen, 1980), and generally believed to be due
to B vitamin deficiencies (Victor, 1975).
The optic nerve has also been used as a system
of choice to study the pre- and postnatal influence
of exposure to a wide range of other teratogenic
agents: X-irradiation (Colello and Schwab, 1994;
Colello et al., 1994), for example, appears to act
primarily by preventing myelin formation in the
retinofugal pathway. Cycloheximide also interferes with myelination in the CNS, possibly by
inhibiting the synthesis of myelin basic protein,
thus altering the ability of oligodendrocytes to
incorporate membrane components into the CNS
myelin sheath (Cullen and Webster, 1989). This
contrasts with the proposed mode of action of
5-azacytidine (5-AZ) which it is believed may
exert its teratogenic effect by altering gliogenesis
in the rat optic nerve (Black and Waxman, 1986;
Black et al., 1986). Similar findings were reported
following prenatal exposure of rats to cortisol
(Bohn and Friedrich, 1982).
The mode of action of teratogens, such as
alcohol, on the optic nerve is likely to be
extremely complex (see above), and is almost
certain to depend on the timing of exposure and
dose involved as well as the biochemical nature
(i.e. molecular configuration) and properties of the
agent under consideration, and may even depend
in certain cases on its mode of administration.
Gearly, there is still much to be learned concerning the mode of action of ethanol. It is only by
pursuing these studies that we may hope to gain
insight into the pathogenesis of this agent's
effects.
2. OBSERVATIONS ON THE POTENTIAL
HAZARD OF EXPOSURE OF PREOVULATORY (UNFERTILIZED) OOCYTES
TO ALCOHOL
It has long been recognized that ~ 50-60% of
all human spontaneous abortuses have an abnormal karyotype, and that of these about 70% are
aneuploid (these are mostly autosomal trisomics
with usually one or two more chromosomes than
the normal complement, while a significantly
smaller proportion are X monosomics with an
XO sex chromosome constitution); a further
20-25% are polyploid, with a triploid: tetraploid
ratio of about 3:1 (Boue" and Boue", 1973;
Therkelsen et al., 1973). These early findings
have been confirmed in all subsequent studies
(see, for example, Warburton et al., 1991).
Most conceptuses with an abnormal karyotype
are aborted during the first trimester of pregnancy,
though very occasionally fetuses with an abnormal
karyotype may survive to later stages of gestation,
or even to term (Ford, 1975; Epstein, 1986), the
most commonly encountered of these neonates
being individuals which are trisomic for either
chromosome 21, 18 or 13, all of whom, if they
survive, show a moderate to severe degree of
mental retardation and reduced life expectancy.
Embryos with autosomal monosomy (i.e. when
only one instead of a pair of autosomes is present),
and those with a Y0 sex chromosome constitution,
do not usually survive much beyond implantation.
Various experimental studies indicate that the
origin of the polyploids (which have multiples of
the normal haploid complement) is most likely to
be due to the fertilization of postovulatory aged
oocytes (Kaufman, 1991, 1995), though this is not
usually associated with an increased incidence of
chromosome segregation errors (O'Neill and
Kaufman, 1988); no age-related effect is observed
in this group. The origin of the aneuploids,
however, appears to be less well understood.
While a relationship clearly exists between
maternal age and non-disjunction, the hypotheses
that have been proposed to account for this have
so far been found to be wanting when tested in
experimental animals (see, for example, Henderson and Edwards, 1968; Luthardt et al., 1973;
Polani and Jagiello, 1976; Speed, 1977).
Indirect evidence from experimental studies
with rodents suggests that exposure to spindle-
TERAT0GEN1C EFFECTS OF ETHANOL
active substances (or trisomigens) that interfere
with the normal functioning of the meiotic
(usually) and, but to a lesser extent, the mitotic
apparatus at the time of fertilization or during
early cleavage, may induce chromosome segregation errors. If exposure to these substances occurs
during the menstrual cycle leading to ovulation,
then the ovulated oocyte may as a consequence
contain an abnormal chromosome constitution
with either one or two more or less chromosomes
than the normal complement — a condition
termed aneuploidy.
a. General observations on possible mechanisms
involved in the induction of aneuploidy
Information regarding the factors that are
known to increase the incidence of aneuploidy is
remarkably limited (Bond and Chandley, 1983;
Epstein, 1986; Dyban and Baranov, 1987; Warburton et al., 1991). While it is clear that there is a
relationship between increased maternal age and
the incidence of non-disjunction, the underlying
mechanism involved has yet to be established.
Much has been written about the relationship
between maternal age and the birth of infants with
Down's syndrome (trisomy 21) (Penrose, 1933;
Penrose and Smith, 1966). While the overall
incidence of this condition is somewhere in the
region of 1 in 2500 of all livebirths, it has been
established that the risk in women over the age of
40 is closer to 1 in 20-40, even though possibly up
to 65-80% of conceptuses with trisomy 21 are lost
at some stage before birth (Creasy and Crolla,
1974; Boue" et al., 1981). In the case of D- and Etrisomics, the prenatal losses may be >90%
(Alberman and Creasy, 1977).
Even though a similar detrimental effect of
maternal ageing appears to be a general phenomenon in mammals, this cannot be the complete
story. Down's syndrome, for example, shows a
bimodal maternal age distribution; one component
(~40% of the affected subjects) belongs to the
age-dependent group (mean maternal age = 43
years) and the others (~60% of all affected
subjects) comprise the age-independent group
(mean maternal age = 28.5 years), where the
mean maternal age corresponds to the peak for
all births in the population. It has long been
recognized that a bimodal distribution with regard
to maternal age is also observed in relation to all
the other trisomies (Warburton et al., 1991).
119
b. When does the malsegregation event occur?
Much useful information is now available from
the cytogenetic analysis of infants with Down's
syndrome in which the parental origin of the extra
chromosome 21 has been unequivocally established (Mikkelsen et al., 1976, 1980; Magenis et
al., 1977) and from the analysis of infants with sex
chromosome anomalies (Race and Sanger, 1969;
Sanger et al., 1971a, b). These studies have
revealed that, in 77% of cases, the malsegregation
event must have occurred in the egg, with
malsegregation at meiosis I and II respectively
accounting for 66% and 11% of these cases, with
the fertilizing sperm being responsible in only
23% of cases (Mikkelsen et al., 1980). In a high
proportion of aneuploid conceptuses, the egg,
when ovulated, must already have had an extra
chromosome present in its complement (Wramsby
et al., 1987). In individuals with a 47.XXY sex
chromosome constitution, the extra X chromosome present is maternally derived in 67% and
paternally derived in 33% of cases, with the
majority of chromosome segregation errors arising
during the first meiotic division (Race and Sanger,
1969; Sanger et al., 1971a, b; see also Angell et
al., 1983, for cytogenetic analysis of human
embryos after in-vitro fertilization).
c. What mechanism might interfere with
chromosome segregation during the first meiotic
division ?
Chromosome malsegregation, whether during
meiotic or mitotic divisions, can only occur as a
consequence of interference with the normal
functioning of either the microtubules or microfilaments of the spindle apparatus, or the centromere (kinetochore) region by which the
chromosomes are attached to the spindle elements,
and which disjoin during anaphase (Mclntosh and
Landis, 1971; Bond and Chandley, 1983). The
cytoskeletal elements may, of course, be polymerized by exposure to a wide range of spindleactive agents with actions on the spindle apparatus
similar to those produced by exposure to substances such as colchicine, Colcemid or cytochalasin.
Following exposure to alcohol, some of the
microtubules at the periphery of the spindle during
anaphase may be displaced from the main body of
the spindle apparatus; multipolar spindles are also
120
M. H. KAUFMAN
commonly encountered, and may be associated
with disorganized alignment of the chromosomes
(O'Neill and Kaufman, 1989). Chromosome 'lagging' is also commonly seen (O'Neill et al., 1989).
The underlying effect of these agents might be
through their capacity to disrupt the regulation of
the changes that normally occur in the concentration of calcium ions in the proximity of the spindle
apparatus during cytokinesis. Certainly there are
many reports which indicate that acetaldehyde
interferes with microtubule integrity and tubulin
polymerization (Tuma and Sorrell, 1987; Tuma et
al., 1991), and it is possible that this effect on
tubulin may account for the induction of chromosome segregation errors observed following exposure to alcohol and other spindle-active substances
(Barthelmess, 1970).
d. Are there any spindle-active substances to
which females are commonly exposed that should
be considered as possible causative agents in
those cases of spontaneous abortion in which a
chromosome malsegregation event has occurred
where no other reasonable explanation is
available?
It is particularly relevant to ask whether there
are any substances that have spindle-active properties to which females might be exposed during
their reproductive life that could account for those
cases of spontaneous abortion with chromosome
segregation errors that cannot be accounted for by
any other known mechanism, such as increased
maternal age or the presence of a balanced
chromosome translocation in the karyotype of
one or other parent.
Exposure to alcohol and/or general anaesthetics,
or possibly other agents that have a similar action
to them on the components of the spindle
apparatus, might be the causative agents in those
cases of spontaneous abortion in which chromosome malsegregation occurs where no other
reasonable explanation is available (Kaufman,
1985; Kaufman and O'Neill, 1988). Both alcohol
and general anaesthetics, even in relatively low
dosage, are spindle poisons, and are certainly
capable of interfering with normal cell division
and chromosome segregation in particular (Kaufman, 1977).
e. Observations in relation to the teratogenic
effect of alcohol
Numerous experimental studies have confirmed
the long-known clinical and epidemiological
observations that alcohol is teratogenic. When
exposure is at high levels, particularly during the
early stages of pregnancy, this can result in the
birth of infants with FAS (for references, see
section 1).
It is at present unclear whether ethanol per se,
or its primary metabolite, acetaldehyde, is the
active teratogen, since both are capable of
inducing a wide range of congenital abnormalities
if given to pregnant experimental animals at
appropriate stages of gestation (for references,
see section 1). When pregnant animals are
exposed to one or other of these agents during
embryogenesis or during early organogenesis, the
cytoskeletal disruption is principally confined to
those cells which are undergoing morphogenetic
shape changes. The neuroepithelial cells, for
example, tend to round up, and the resultant
interference that occurs with the process of
neurulation produces neural tube defects along
the embryonic axis (O'Shea and Kaufman, 1981).
Similarly, cellular disruption can occur during
early cardiogenesis, with the production of cardiac
defects (O'Shea and Kaufman, 1981).
/ Clinical findings with regard to the influence
of alcohol on the chromosome constitution of
individuals
Occasionally infants with FAS have an abnormal karyotype (e.g. Qazi et al., 1978; Gardner et
al., 1985; Bingol et al., 1987); in each of these
published examples, different chromosomes were
involved. In one of these studies (Bingol et al.,
1987), five cases of infants with FAS associated
with trisomy 21 were described. In these infants,
growth deficiency was more pronounced than with
FAS alone, and all had congenital heart disease.
Trisomy 21 and FAS may occur together, at
random, in 1 in 525 000 newborn infants in the
USA. In this study, the incidence was 1 in 6600, a
30-fold increase over the chance occurrence of
these two conditions together. All of these cases
had a chronic alcoholic mother as well as maternal
grandmothers, and it was suggested that there
might be an increased incidence of trisomy 21 in
children with two generations of alcoholic
TERATOGENIC EFFECTS OF ETHANOL
mothers.
This situation can only arise, assuming both
parents have a normal chromosome constitution, if
one or other gamete is exposed to a mutagenic
stimulus (in this instance, exposure may have
occurred to alcohol or its breakdown product
acetaldehyde, which is known to be mutagenic)
during gametogenesis or at the time of conception.
It is known that acetaldehyde, for example, can
interfere with the functioning of the mitotic/
meiotic spindle apparatus, inducing chromosomal
aberrations, sister chromatid exchanges and crosslinks between DNA strands (Obe and Ristow,
1979; Natarajan and Obe, 1982).
Chromosome aberrations are also seen in
alcoholics (De Torok, 1972), and the underlying
mechanism may be due to interference by alcohol
or acetaldehyde with the functioning of the spindle
apparatus (see above). The high degree of
constancy of the normal diploid karyotype
among all age groups of the nonalcoholic (control)
somatic cells is contrasted by the non-specific and
unpredictable nature of the cells of the alcoholic
subjects, particularly in those with alcoholconnected organic-brain-syndrome. From these
patients, 43.7% of the cells had a 2n—1 chromosomal complement, with other cells having 2n—2,
2n + l and 2n—3 complements, with still many
other secondary and minor modes including
changes in chromosome structure. In some subjects, only 4.4% of the counted cells carried a
normal diploid complement of 46 chromosomes.
In the FAS infants with an abnormal karyotype
(see above), all of their cells have an identical
chromosomal aberration. This contrasts with the
situation seen in alcoholics where the majority of
their cells have an aberrant and variable chromosome constitution.
g. The findings from experimental studies used to
test the hypothesis that exposure of pre-ovulatory
oocytes to ethanol is capable of inducing
aneuploidy
When ovulated unfertilized mouse eggs are
briefly exposed in vitro to a dilute solution of
ethanol in tissue culture medium, very high rates
of parthenogenetic activation are achieved.
Parthenogenesis is defined as the production of
an embyro, with or without eventual development
into an adult form, from a female gamete in the
absence of any contribution from a male gamete.
121
In mammals, this is largely an experimental
phenomenon, but it is commonly employed as a
reproductive strategy in many forms of animals
and plants (for details, see Kaufman, 1983a).
Cytogenetic analysis of the first cleavage mitosis
of these embryos revealed that between 10 and
20% of the preparations examined displayed
evidence of non-disjunction. In ~80% of the
latter cases, either one extra chromosome was
present or one missing from the expected
complement. In the remaining 20% of this
group, two extra chromosomes were involved in
the malsegregation event (Kaufman, 1982). No
evidence was found that specific chromosomes
were more likely to be involved in these
malsegregation events than other members of the
complement (O'Neill and Kaufman, 1989).
Parthenogenetic activation per se is not normally
associated with non-disjunction, and it is the
properties of the agent employed to induce this
phenomenon that appear to be the critical factor
(Kaufman, 1983a; O'Neill and Kaufman, 1989).
In a follow-up study, recently mated female
mice were given a dilute solution of ethanol by
mouth, and the chromosome constitution of the
fertilized embryos determined at the first cleavage
mitosis. Male mice bearing easily recognizable
balanced translocation 'marker' chromosomes
were used in order to establish whether the
malsegregation event involved the oocyte- or
sperm-derived chromosome set. In all instances
where there was evidence of non-disjunction, this
involved the oocyte-derived set of chromosomes
(Kaufman, 19836). Since both series of experiments involved the exposure of oocytes or recently
fertilized embryos to ethanol during the time that
they were completing the second meiotic division,
subsequent studies were carried out to investigate
whether exposure to similar levels of ethanol in
vivo given during the completion of the first
meiotic division produced a similar or different
result (Kaufman and Bain, 1984). Cytogenetic
analysis revealed that any non-disjunction
observed was exclusively confined to the oocytederived chromosome set. Complementary studies
in which unfertilized oocytes and very recently
fertilized 1-cell stage embryos were exposed in
vivo to the general anaesthetic Avertin produced
almost identical results (Kaufman, 1977; Kaufman
and Bain, 1984).
These experimental findings have confirmed
122
M. H. KAUFMAN
that exposure of unfertilized mouse oocytes to
The author's experimental findings indicated
spindle-active agents during the first meiotic that the potential hazard of exposure of predivision can lead to non-disjunction. Since alcohol ovulatory human eggs and, but to a lesser extent,
is the only spindle-active agent which is freely recently fertilized embryos, to alcohol is at least as
available to women during their reproductive harmful as exposure to this agent during preglives, these observations would seem to indicate nancy, and consequently this should be an equal
that it is the most likely causative agent in those cause for concern. This specific topic was
spontaneous abortions with aneuploidy where no discussed in the House of Lords almost 10 years
other factor is involved (Kaufman, 1984, 1985; ago (Whaddon, 1987), but little information in this
Kaufman and O'Neill, 1988).
regard has apparently percolated down into the
It has been stated that the risk of spontaneous public consciousness in the interim years. The
abortion is twice as high in women drinking 1 oz. availability of such information should by now be
of absolute alcohol as infrequently as twice per within the public domain, and certainly should be
week compared to matched controls (see Kline et readily available to medical practitioners and
al., 1980). These latter authors noted that it was ancillary workers whose role it is to advise
unclear whether this was due to a fetotoxic rather individuals who wish to maximize their chance
than a teratogenic effect. If it is assumed that the of having a successful outcome to their pregnancy.
alcohol was just as likely to have been consumed
before as after the onset of pregnancy, then the Acknowledgements — I particularly wish to thank Drs K. S.
effect could just as easily have been mutagenic on O'Shea, G. T. O'Neill, S. H. Parson and Y. Y. Dangata who
the mother's germ cells. Mutagenicity tests at various times have shared the author's interest in the
teratology of alcohol, and whose findings are discussed
indicate that ethanol, both by itself but principally here.
via its metabolite acetaldehyde, is mutagenic in
both male rodents (Badr and Badr, 1975; Barilyak Note. In a field of study as extensive as this, the publiand Kozachuk, 1981; Hunt, 1987) and female cations cited should only be viewed as representative rather
rodents (Alvarez et al., 1980), and is equally likely than exhaustive, and serve as a guide for those who may
to be so in the human should exposure occur wish to pursue individual topics considered in this review in
greater detail.
during appropriate stages of gametogenesis.
GENERAL CONCLUSIONS
Despite the fact that the CNS in the prenatal and
early postnatal period, and the optic nerve in
particular, is extremely sensitive to a wide range
of teratogenic stimuli, it is its sensitivity to ethanol
which should be the greatest cause for concern: (i)
because of its unrestricted availability; (ii) its
universal acceptance as a 'harmless stimulant'.
While the risk of exposure to alcohol during
pregnancy is now fully accepted in 'informed'
circles, to date, no appropriate legislation has
appeared in the UK, as is in force in many other
countries where wine and spirits are required to
have a warning notice located in a prominent place
on the label, which draws attention to the potential
risk of damage to the fetus if exposure to alcohol
occurs during early pregnancy. This would be
equivalent to the Chief Medical Officers' health
warning at present displayed prominently on
cigarette advertising and on all packets of cigarettes sold in the UK and in many other countries.
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