5.

03 Embryotoxicity: Anatomical, Physiological, Functionalq

JM DeSesso, Exponent, Inc., Alexandria, VA, United States; and Georgetown University School of Medicine, Washington, DC, United
States
AL Williams, Exponent, Inc., Alexandria, VA, United States
© 2018 Elsevier Ltd. All rights reserved.

5.03.1 Introduction 21
5.03.1.1 Rationale for Extrapolating Animal Results to Humans 21
5.03.1.2 Events in Early Development 23
5.03.1.3 Embryonic Potency and Differentiation 23
5.03.2 Case Study: Anatomical Changes 24
5.03.2.1 Inorganic Arsenic 24
5.03.2.2 Critical (Sensitive) Period for Teratogenesis 25
5.03.2.3 Response Threshold 26
5.03.3 Case Study: Physiological Changes 26
5.03.3.1 Hemoglobin-Based Oxygen Carriers and Yolk Sac Poisoning 27
5.03.3.2 Inverted Yolk Sac Placentae in Short-Gestation Species 27
5.03.4 Case Studies: Functional Changes 29
5.03.4.1 Cadmium and the Developing Lung 29
5.03.4.2 Mercury and Fluorine and the Developing Kidney 30
5.03.4.3 Autism and Autism Spectrum Disorder 30
5.03.5 Conclusion 31
References 31
Further Reading 33

5.03.1 Introduction

Mammalian embryos seem relatively defenseless, but are surprisingly resilient organisms. While embryos can survive many
challenges, if these challenges arise during sensitive periods of embryogenesis, the resulting term offspring may be abnormal
and may experience a shortened lifespan. Among humans, birth defects remain an important adverse health consequence despite
many years of research investigating their causes and potential ameliorative strategies. While the financial and emotional impacts of
birth defects on individual families are enormous, the scale of the problem is not appreciated by the general public. According to the
National Center for Health Statistics (Hamilton et al., 2015), there were 3,988,076 births in the United States in 2014, which equa-
tes to the birth of a baby approximately every 7.4 s. According to the March of Dimes (2009), one of every eight babies is delivered
preterm; a preterm or low birth weight babies is born every 2 min; and a baby with a birth defect is born every 4 1/2 min. Table 1
presents the leading causes of infant mortality to the age of 1 year in the United States during 2012 (Heron, 2015). The table reveals
that approximately one in every five infant deaths is a consequence of birth defects and that the leading causes of nearly one half of
all infant deaths are related to birth defects, low birth weight, or complications of pregnancy.
The prevalence of adverse pregnancy outcomes in humans is presented in Table 2 (adapted from Schardein, 1993). Inspection of
the table reveals that less than 20%–30% of human zygotes (fertilized ova) and less than half of all pregnancies (zygotes that
implant into the uterine wall) result in normal, healthy babies. Thus, the early life of the embryo is a period of vulnerability
and one which cannot be studied directly in humans for obvious ethical reasons. Animal models, particularly rodents and rabbits,
have provided much of the knowledge that has been used both to understand how exogenous agents interact with developing
embryos and to serve as surrogates for safety testing.

5.03.1.1 Rationale for Extrapolating Animal Results to Humans

The endpoints of concern and developmental toxicity studies are measured in the offspring and include:

q
Change History: January 2017. Dr. AL Williams updated Tables 1 and 2 in the Introduction (1.0) and 4 (3.2) as well as the texts that described them. Dr. JM
DeSesso updated the discussion of Inverted Yolk Sac Placenta and HBOC experiments (3.2). Both authors contributed to general rewording throughout the
chapter, proofreading the chapter, and updating the references (both adding and deleting references). The figures were redrawn and proofed under the
direction of both authors.
This is an update of JM DeSesso, Embryotoxicity: Anatomical, Physiological, and Functional, Comprehensive Toxicology, Second Edition, edited by Charlene
A. McQueen, Elsevier, Oxford, 2010, Volume 12, Pages 11–25.

21
22 Embryotoxicity: Anatomical, Physiological, Functional

Table 1 Leading causes of infant mortality in the United States in 2012 (to age 1 year), total births: 3,952,841

Cause of death Number Percent of deaths Rate (per 100,000)

All causes 23,629 100 597.8

a
Birth defects 4939 20.9 124.9
a
Preterm/low birth weight 4202 17.8 106.3
Sudden infant death (SIDS) 1679 7.1 42.5
a
Pregnancy complications (\) 1507 6.4 38.1
Accidents 1169 4.9 29.6
a
Complications of placenta/cord 1018 4.3 25.8
Sepsis of newborn 566 2.4 14.3
Respiratory distress (RDS) 504 2.1 12.8
Disease of circulatory system 492 2.1 12.4
Neonatal hemorrhage 422 1.8 10.7
a
Causes related to development and pregnancy.
Sources: Heron (2015) and Martin et al. (2013).

Table 2 Adverse pregnancy outcomes in humans

Preimplantation loss 40%–60%

After implantation
Miscarriage (postimplantation loss) 10%–15%
Major birth defects 3%
Tardive effects
By 2 years of age 6%–7%
By 8 years of age 8%
Minor birth defects 11%
Low birth weight 8.0%
Infant mortality 0.6%
Preterm birth 11.4%

Jones and Lopez (2014), March of Dimes (2015), and Schardein (1993).

l embryo or fetal death,

l structural changes,
l growth alterations, and
l functional deficits.

The first three of the endpoints are assessed by investigators at the time of study termination. Death of the offspring and gross
changes in body structure are readily determined by observations. Growth alterations are assessed by weighing fetuses. Functional
deficits are usually not determined by development toxicity tests, although they can be assessed as part of a multigeneration repro-
duction study or in a neurobehavioral developmental study. The advent of the complex study design of the extended one-generation
reproductive toxicity study has provided increased options for evaluating various postnatal effects when these types of effects are
suspected based on findings in the early phase of the study.
A primary concern of toxicologists has been the terminology used to describe structural changes in the offspring. While standard-
ized names of fetal observations have been developed (Wise et al., 1997) and are being improved (Paumgartten et al., 2009), defi-
nitions have not been agreed upon for reporting the severity of serious versus minor structural changes. Because the term
“malformation” implies the presence of devastating consequences in offspring when exposure of pregnant animals to their products
during safety pharmacology tests and the threat of categorization as a reproductive hazard in the European Union, a rich lexicon has
evolved to connote changes in fetal structure. These terms attempt to convey information without the emotional or sensational
impact of “malformation.” A list of terms that have been used to describe structural alterations in fetuses is presented in Table 3.
It is important to understand the criteria that have been used to decide whether morphological changes are of sufficient magni-
tude to warrant being called out in the results of developmental toxicity studies. The four major observational determinants include
the degree of deviation from the average state, the rarity of occurrence for a particular observation, the impact of the observed change
on the presumed health status of the fetus, and the cosmetic significance of the finding. There is a degree of subjectivity in the assess-
ment of the observational determinants, especially with regard to cosmetic significance and minor impacts on health. If one were to
compare the knowledgebase concerning the findings of experimental animal studies compared to those of human epidemiology
studies, there are some significant differences. On the negative side, epidemiology studies are less rigorously monitored than animal
studies, and the human population has far greater phenotypic heterogeneity than experimental animals. Because many epidemi-
ology studies are retrospective nature, they are plagued with recall bias and less amenable to the dose–response evaluations
than animal studies. On the positive side, the ramifications of observations in human fetuses are far better understood than those
 Embryotoxicity: Anatomical, Physiological, Functional 23

Table 3 Descriptive terms used to describe fetal observations

l Developmental deviation
l Structural change
l Malformation
l Anomaly
l Congenital defect
l Anatomical alteration
l Terata
l Structural aberration
l Deformation
l Variation (minor changes)

in animals. For instance, humans do not understand the potential impact of the loss of a single vibrissa to a rat pup; whereas the
consequence of a cleft nose in a human infant is readily understood as an impediment to a normal social life as an adult.
Suffice it to say that all objective terminology should be directed toward describing the results of altered development in the
embryo. Consequently, it is important to have some understanding of the development of embryos during the first one-third to
one-half of gestation. The following paragraphs will briefly describe the events that occur during early gestation in order to form
a foundation for understanding how exogenous agents might perturb development and when these perturbations should be consid-
ered serious.

5.03.1.2 Events in Early Development

In mammals, fertilization of an ovum by a single spermatozoon occurs in the uterine tube, resulting in the creation of a zygote with
the diploid complement of chromosomes. Fertilization determines the sex of the zygote and initiates a rapid series of mitotic
divisions known as cleavage divisions, which divides the copious cytoplasm of the ovum into progressively smaller cells. Cleavage
divisions take place while the zygote is transported down the uterine to the uterine cavity, eventually converting the organism from
a large unicellular zygote to a cluster of small cells known as a morula. The morula soon acquires a lumen and is henceforth known
as a blastocyst. The blastocyst comprises a sphere of cells (trophoblast) that will give rise to the fetal membranes and placenta; and
a cluster of cells on the interior, known as the inner cell mass, which will give rise to the embryo proper. The trophoblast layer of the
blastocyst contacts, adheres to, and becomes intimately associated with the epithelium of the uterine wall where it will develop
a mechanism for transfer of nutrients and oxygen from the maternal system to that of the offspring. The temporary organ that is
developed from tissues of the maternal organism and her offspring is the placenta. Details regarding placental morphology and
function are discussed elsewhere (e.g., Ramsey, 1982; Kaufman ad Burton, 1994; Carney et al., 2004; DeSesso et al., 2012).
Development of an organism from zygote, to embryo, to an independent being requires the coordination of numerous simul-
taneous processes in specific sequences that take place at precise times during gestation and postnatal life. This is especially true for
rodents, whose brief gestations result in conditions such that some organ systems of rodent neonates have attained only the state of
maturation found in late second or early third trimester human fetuses, whereas other organ systems are fully functional (Morford
et al., 2004). While details concerning the development of embryos from various species (e.g., length of gestation, size of fetuses,
time at which developmental landmarks appear) may differ, the sequence of developmental events and many other features and
processes are remarkably consistent across species.
The inner cell mass promptly segregates into a two-layered disk: one layer (epiblast) is associated with the developing amniotic
cavity; the other layer (hypoblast) is associated with the developing yolk sac cavity. The epiblast undergoes a process of cellular
migration (variously called gastrulation, ingression, or invagination; Browder et al., 1991; Gilbert, 1997; Carlson, 2014; Schoenwolf
et al., 2008) to rearrange into the three germ layers (ectoderm, mesoderm, and endoderm). Specific tissues are derived from each
germ layer such that the ectoderm gives rise to the nervous system and skin; mesodermally derived tissues include cartilage, bone,
muscle, connective tissue, kidneys, and blood; and the endoderm gives rise to the linings of the alimentary, respiratory, and lower
urinary tracts. The primordia of the organ systems are formed from combinations of tissues derived from the germ layers. To execute
this process efficiently and accurately, many controls operate to maintain embryonic schedules and to control the fates of popula-
tions of cells.

5.03.1.3 Embryonic Potency and Differentiation

To help understand how the development of the embryo proper unfolds in an orderly fashion, two important concepts will be
explained: potencydthe potential fate of a cell at any given time in development; and differentiationdthe process whereby cells attain
the intrinsic properties and functions of a particular adult tissue.
Early in development the embryonic cells are pluripotent (i.e., they have the ability to develop into a great number of different
tissue types). As development proceeds, they differentiate (i.e., become committed to a particular structural and/or functional role
within the body). This concept of increasing cellular/organ differentiation, or specialization, is illustrated diagrammatically in
24 Embryotoxicity: Anatomical, Physiological, Functional

Fig. 1 A conceptualized synopsis of embryonic development. The flow of time is from left to right. The developmental decisions of tissues as they
differentiate are designated by diverging arrows. Note that early differentiation coincides with the time of implantation. Susceptibility to developmen-
tally toxic agents is greatest during the periods of early differentiation and organogenesis, when the greatest number of downstream arrows can be
affected by an interruption at a single decision point. Note that as time elapses and the embryo ages, developmental decisions are made, and early
tissues differentiate. Very early cells (e.g., cells of the inner cell mass) are morphologically similar and have the potential to become nearly any type
of embryonic tissue. As the cells increase in differentiation, their embryonic potency decreases and their ability to develop into adult tissues is
restricted to a narrower set of choices. DeSesso, J. M. (2012) “Comparative Gestational Milestones in Vertebrate Development,” Chapter 6 in Devel-
opmental and Reproductive Toxicology: A Practical Approach, 3rd Ed., R. D. Hood, Ed., Informa Healthcare, London, pp. 93–138.

Fig. 1. This process continues throughout development and occurs in all species, although the chronological timetables can be very
different from species to species. The developmental time course for species with longer gestation periods (e.g., humans) occurs over
a greater period of time than that of species with shorter gestations (e.g., rodents). Thus, comparative embryonic schedules for
humans and animals used in experimental studies have been compiled by a variety of authors (e.g., Nelsen, 1953; Otis and Brent,
1954; Edwards, 1968: Hoar, 1976; Hoar and Monie, 1981; DeSesso, 1997, 2006, 2012).
That interspecies differences exist not only with respect to embryonic susceptibility to developmental toxicants but also
regarding various aspects of gestational physiology is well established. This situation makes the assessment of the risk of develop-
mental toxicity from results observed in animal experiments a challenge. Despite the interspecies differences in the responses to
developmental toxicants, the process of embryonic development in species as diverse as round worms, fruit flies, fish, amphibians,
and mammals proceeds through a coordinated series of activities that encompasses induction, specification of cell fate, differenti-
ation, morphogenetic cell movements, and apoptosis. Furthermore, these activities are controlled by developmental mechanisms
that are highly conserved at the molecular level. For instance, approximately 17 signaling pathways have been identified in verte-
brate species (NRC, 2000). Regardless of what species is under study, these signaling pathways are vulnerable to disruption provided
that the concentrations of a given toxicant exceed the response threshold. It is this underlying, shared commonality that provides the
scientific underpinning for the use of animal models for risk assessment.

5.03.2 Case Study: Anatomical Changes

5.03.2.1 Inorganic Arsenic
Inorganic arsenic in its various forms has been used by many investigators as a model compound to study the pathogenesis and
potential mechanisms of exencephaly, the homolog of anencephaly seen in humans (e.g., Ferm and Carpenter, 1968; Hood,
1972; Hood and Bishop, 1972; Beaudoin, 1974; Umpierre, 1981; Willhite, 1981; Hood and Harrison, 1982; Carpenter, 1987).
Investigations in animal models have shown that inorganic arsenic readily crosses the placenta (Hanlon and Ferm, 1977; Dencker
 Embryotoxicity: Anatomical, Physiological, Functional 25

et al., 1983; Lindgren et al., 1984; Hood et al., 1987, 1988). Direct exposure to inorganic arsenic using whole embryo culture of
rodent embryos has revealed inorganic arsenic to be developmentally toxic (e.g., Chaineau et al., 1990): the exposed rodent
embryos exhibit reproducible subcellular and metabolic changes including expression of stress proteins (Mirkes and Cornel,
1992), increased methylation of DNA with subsequent alterations in gene expression (Wlodarczyk et al., 1996a,b), and possible
formation of oxygen-free radicals (Tabacova et al., 1994a,b). Findings such as these have been interpreted to demonstrate an asso-
ciation between exposure of pregnant mammals to inorganic arsenic and neural tube defects (e.g., Barlow and Sullivan, 1982;
Schardein, 1993; Golub, 1994; Shepard, 1995; Shalat et al., 1996).
This case study illustrates the point that to elicit anencephaly or exencephaly, inorganic arsenic must attain a threshold concen-
tration in the embryo and that this must happen during the susceptible period in development. For some substances, when expo-
sure occurs by typical routes (e.g., oral or inhalation), malformations may not be seen because the threshold concentration of
inorganic arsenic required to elicit the malformation in the embryo may be higher than that which will cause the pregnant female
to suffer severe toxicity, including death.
The best information for determining potential human developmental risk comes from well-designed epidemiology investiga-
tions that study populations exposed to known amounts of arsenic. In the case if inorganic arsenic, good epidemiology data are not
available. Most of the extant epidemiology studies are ecologic investigations of people who drank water containing arsenic (Zierler
et al., 1988; Aschengrau et al., 1989; Börzsönyi et al., 1992) or those who lived near either smelters (Nordström et al., 1978a,b,
1979a,b; Beckman and Nordström, 1982; Tabacova et al., 1994a,b) or industrial facilities that processed large amounts of arsenic
(Ihrig, 1997; Ihrig et al., 1998). In general, these studies did not verify or quantitate arsenic exposures during pregnancy; they did not
account for potential confounding factors such as smoking and alcohol habits, maternal age, and socioeconomic/nutritional status;
and the investigations focused on populations who were exposed to ill-defined mixtures of materials, which may have included
inorganic arsenic (Jacobson et al., 1999). These and other flaws make the human data set inadequate (Golub et al., 1998; NRC,
1999). Nevertheless, experimental evidence suggests that if inorganic arsenic were to reach the human embryo under the appro-
priate conditions, adverse morphology would result.
As mentioned previously, the potency and its state of differentiation of embryonic cells are reciprocal characteristics. During early
stages of development, embryonic cells are morphologically and biochemically similar, and they have the potential to become
nearly any type of cell in the body. As development proceeds, however, developmental decisions are made concerning the fate
of each cell. Thus, at later periods of gestation groups of cells have become dissimilar and will respond differently to environmental
challenges. This means that as the embryo grows and matures, the population of cells that is susceptible to a toxicant will comprise
a relatively smaller amount of the embryo. This is the likely explanation for the phenomenon that during the time when organs are
first laid down (organogenesis), most organs undergo their “critical period” during which they are most susceptible to damage by
environmental agents. As gestation proceeds and the tissues of a developing organism become more differentiated, the organism’s
susceptibility to grossly observed developmental toxicity tends to decrease. This led Wilson (1959, 1973) to generalize that the time
at which an agent acts on an embryo determines which tissues will be susceptible to the effects of the agent and consequently that
susceptibility to a particular agent varies during gestation. Interestingly, exposure to toxicants, even at high doses, during the period
from fertilization through formation of the blastocyst typically does not cause malformations. The apparent resistance of young
embryos is probably due to the fact that the cells of the zygote have not begun to differentiate and therefore they are still pluripotent,
which allows the embryo to replace any affected cells, thereby permitting normal development to proceed. Although destruction of
a number of undifferentiated cells may not result in a malformation, there does appear to be a critical limit beyond which damaging
even nonspecialized cells cannot be tolerated if the embryo is to survive; if that critical limit is exceeded, the zygote will die.

5.03.2.2 Critical (Sensitive) Period for Teratogenesis

During the period of early organogenesis (when the embryo begins to undergo differentiation and the establishment of the germ
layers), the onset of greatest susceptibility to teratogenesis occurs (see Fig. 2). The onset of susceptibility to teratogenesis is sudden
and the majority of teratogenic agents produce their highest incidences of malformations at about this time. Not only do embryos
themselves have a sudden onset of susceptibility to teratogenesis, but also each organ of an embryo has a sensitive period for tera-
togenesis (Wilson, 1973; DeSesso and Harris, 1996). This sensitive (or critical) period coincides with the early developmental events
and tissue interactions that occur within the organ. In general, the susceptibility to teratogenesis decreases as differentiation and
organogenesis proceed. As an embryo progresses through organogenesis, and as differentiation continues, production of a given
teratogenic effect requires increasingly higher doses of a teratogen. This means that as organ systems and the embryo itself become
progressively more differentiated, they become increasingly resistant to teratogenesis. For an effect to be manifest in an organ rudi-
ment, the concentration of the toxicant at the site within the embryo must exceed a threshold. Below the threshold concentration,
no gross malformations will be induced.
By the time of late organogenesis and the early fetal period, most organ systems have been laid down and the critical events
involved in their formation have been completed. What remains to be accomplished during the remainder of prenatal and postnatal
development is the progressive growth and functional maturation of each organ system. Consequently, the majority of gross mal-
formations become increasingly less problematic, although malformations of late-developing organs (e.g., kidneys, genitalia,
brain), altered histodifferentiation, growth retardation, and postnatal functional deficits (including neurobehavioral problems)
may be caused following interruption of events during the period of late organogenesis and early fetal life.
26 Embryotoxicity: Anatomical, Physiological, Functional

Fig. 2 This diagram shows idealized curves that portray relative maternal blood concentrations of arsenic versus time after exposure of the dam to
inorganic arsenic by intravenous, intraperitoneal, oral, and inhalational routes. The horizontal dashed line represents the theoretical response threshold
or blood concentration that must be exceeded to elicit a toxic response. DeSesso, J. M. (2012) “Comparative Gestational Milestones in Vertebrate
Development,” Chapter 6 in Developmental and Reproductive Toxicology: A Practical Approach, 3rd Ed., R. D. Hood, Ed., Informa Healthcare, Lon-
don, pp. 93–138.

The rudiment of the nervous system is the first organ system to appear in the embryo (Schoenwolf and Smith, 1990, 2000;
DeSesso et al., 1999). The neural tube closes by gestational day (GD) 9.5–11 in mouse, rat, and rabbit and by GD 28 in humans
(DeSesso, 2006). Thus, the most susceptible period for exposure to an agent that causes exencephaly in mice and rats is GD 8; in
humans, exposure must occur prior to GD 25 for an agent to cause anencephaly. Consequently, embryonic exposure to inorganic
arsenic must occur early in gestation to cause cranial neural tube defects. For the most part, the purpose of the early rodent studies
(prior to 1995) using inorganic arsenic was to investigate the pathogenesis of cranial neural tube defects, which were produced by
injection of maternally toxic (and often nearly fatal) doses of arsenic directly into the veins or abdominal cavities of pregnant
animals (for reviews, see Golub et al., 1998; DeSesso et al., 1998). The investigators understood that their experiments were
designed to yield a high occurrence of neural tube defects, not to assess the risks to humans from environmental exposures to arsenic
(Mottet and Ferm, 1983; Willhite and Ferm, 1984).

5.03.2.3 Response Threshold

In order to determine whether environmental exposures to inorganic arsenic posed a risk of neural tube defects, a series of labora-
tory studies (Nemec et al., 1998; Stump et al., 1999; Holson et al., 1999a,b) using relevant (oral and inhalational) exposure routes
and a dose–response design was conducted in animals commonly used in reproductive toxicity tests (mice, rats, rabbits). None of
the investigations reported arsenic-related structural malformations (including neural tube defects) in fetuses from dams receiving
either oral or inhalation exposures although the high dose levels, which caused maternal toxicity and deaths, did cause low birth
weights and resorptions. Parallel studies using injection of inorganic arsenic into rats and mice did produce neural tube defects.
The results of these studies were integrated with the copious amount of previous experimental data on inorganic arsenic in a risk
assessment of the potential developmental toxicity of inorganic arsenic published by Holson et al. (2000), who also reported the
time course of experimentally measured arsenic concentrations in maternal blood after oral, intraperitoneal, and inhalation (dust)
exposures (Fig. 3). The authors concluded that arsenic-induced malformation in experimental animals depends upon both the dose
and the route of administration of inorganic arsenicals. They suggested that structural malformations are induced only when
maternal blood concentrations of inorganic arsenic exceeded a theoretical threshold that was only exceeded (and compatible
with life) after intraperitoneal injections. Consequently, they concluded that exposure to inorganic arsenic by environmentally rele-
vant routes (oral and inhalation), poses little to no risk of causing structural malformations in offspring unless the exposure is of
sufficient magnitude to cause concomitant, nearly-fatal effects in the mothers. Such maternal effects do not occur at exposure
concentrations normally encountered by people.

5.03.3 Case Study: Physiological Changes

The physiology of embryos is dynamic. As the embryo develops and grows, its requirements for nutrients and gases change. The early
embryo exists in a nearly anaerobic state for the first few days of life. As the embryo implants and the need for oxygen and nutrients
increases, there are modifications in the transport characteristics of the trophoblast and the mitochondria of embryonic cells (Benos,
1981; Shepard et al., 2000; Sathananthan and Trounson, 2000). The milieu in which the embryo resides also changes: the embryo
 Embryotoxicity: Anatomical, Physiological, Functional 27

Fig. 3 A diagram to illustrate the relationship between the relative teratogenic sensitivity of embryos and increasing gestational age. The dashed line
between fertilization and implantation represents the limited number of agents that have been reported to cause effects in term offspring when
administered at this time. Susceptibility increases rapidly after implantation of the blastocyst and peaks during the period of organogenesis. While
susceptibility declines during the late embryonic and fetal periods, alterations in late forming organs and tissues (e.g., genitalia, brain) can occur
during these periods. DeSesso, J. M. (2012) “Comparative Gestational Milestones in Vertebrate Development,” Chapter 6 in Developmental and
Reproductive Toxicology: A Practical Approach, 3rd Ed., R. D. Hood, Ed., Informa Healthcare, London, pp. 93–138.

develops the means for supplying increasing amount of nutrients by means of exchange with the maternal organism through
a placenta: an organ composed of fetal and maternal tissues apposed for physiologic exchange (Mossman, 1937). It also develops
the means to transport the nutrients from the placenta to the embryo through a cardiovascular system of its own.
This case study illustrates a physiologic mechanism that operates in the specialized inverted yolk sac placenta. Inverted yolk sac
placentae are found in rodent and lagomorph embryos, but are of no importance in humans because human embryos do not
develop an inverted yolk sac placenta.

5.03.3.1 Hemoglobin-Based Oxygen Carriers and Yolk Sac Poisoning

Injectable hemoglobin-based oxygen carriers (HBOC), which weigh approximately 250,000 Daltons, possess considerable thera-
peutic potential for replacing red blood cell function in patients suffering from blood loss. An early pilot study that was conducted
in pregnant rats to establish the doses for a definitive developmental toxicity study was confounded by severe maternal toxicity,
precluding assessment of offspring (Stump et al., 2015). In this study groups of eight pregnant rats were infused continuously
throughout organogenesis (GDs 6–18) at doses that were 20 times the maximum expected human dose and equivalent to 10 times
the total blood volume. The large increase in blood volume was the likely reason for the maternal toxicity. A second pilot study
(Stump et al., 2015) used lower amounts of HBOC (1.95–5.85 g/kg/day) infused into groups of six rats on single GDs (i.e.,
GDs 6–7, 7–8, 8–9, 9–10, 10–11, 11–12, or 12–13). In this study, no developmental toxicity was observed in fetuses of the low
dose group, but fetal death and malformations were prominent in the high dose groups exposed on GDs prior to GD 10. This
is not the typical pattern of effects elicited in rats by most developmental toxicants, which usually cause the highest incidence
and diversity of malformations after exposures occurring on GDs 11–13; malformation incidences usually decrease from the
peak on day 13 through GD 15 or 16. Interestingly, as discussed below, rodents and rabbits develop a special type of placenta
that is the primary means for transferring nutrients to the embryo during the period when the developmental toxicity to HBOCs
was observed.

5.03.3.2 Inverted Yolk Sac Placentae in Short-Gestation Species

A clear disparity between humans and the experimental animals used for assessing potential developmental toxicity in safety tests is
the duration of gestation. The gestation periods of the rat, rabbit, and human average 22, 31, and 266 days, respectively (Table 4).
The short gestational periods of rodents and the rabbit lead to compressed developmental schedules compared to that of the
human. All aspects of development must occur much more quickly in these species, including the rapid formation of mechanisms
to exchange nutrients, gases, and waste products with the maternal system. The urgency for developing a nutrient exchange mech-
anism in short-gestation species is amplified by the fact that in most cases over the course of 5–7 days the zygote, which forms in the
uterine tube, develops to the blastocyst stage, enters the uterine cavity, and attaches to the uterine endometrium (Table 4). Ingres-
sion, establishment of the germ layers and major organogenesis (the most sensitive period for developmental toxicity) are
28 Embryotoxicity: Anatomical, Physiological, Functional

Table 4 Schedule of gestational landmarks for various species (in gestational days)

Species Blastocyst in uterine cavity Implantation begins Inverted yolk sac placenta Chorioallantoic placenta Palate closes Gestation length

Mouse 4 5 7 9 15 20
Rat 5 5.5–6 6.5–7 11–11.5 17 22
Rabbit 5 7.5 9 12.5 20 32
Dog 12–16 12–18 NA 22 35 60
Monkey 5–8 9 NA 25 45 166
Human 4–5 7 NA 27 60 266

Sources: DeSesso (2012), Holson et al. (2005), Foote and Carney (2000), and Ramsey (1982).

completed in rodents and rabbits within about 10 days after implantation begins. What is more remarkable is that the definitive
chorioallantoic placenta is not established until approximately GD 9 in the mouse (Theiler, 1972; Downs, 2002), GD 11.5 in
the rat (Jollie, 1990), and GD 12.5 in the rabbit (Foote and Carney, 2000). By this relatively late time in the gestation of these
species, many significant developmental milestones have passed (cf. DeSesso, 2006; 2012). To meet the nutritional and metabolic
needs of the embryo, transport of nutrients and gases during the early postimplantation period in the short-gestation species is
accomplished by means of an early mechanism that is derived from the embryonic yolk sac: the inverted yolk sac placenta.
The mouse, rat, and rabbit all develop an inverted yolk sac placenta, a structure which is never present in humans. Carney et al.
(2004) described the anatomic differences in rat, rabbit, and human placentation, what factors impact transfer of substances
between maternal and offspring compartments, and under what conditions the responses of these species is representative of
a possible mode of action in the human have been described and assessed (Carney et al., 2004; DeSesso et al., 2012). Briefly, in
rodents and the rabbit, after the zygote develops rapidly into a sphere of cells (the blastocyst), the lumen of the sphere is subdivided
by the presence of an inner cell mass (the future embryo) such that the cavity above the inner cell mass will become the amniotic
cavity and the one below the inner cell mass will become the yolk sac cavity.
The yolk sac cavity is initially much larger than the amniotic cavity and growth of the inner cell mass results in the yolk sac cavity
taking on a configuration much like that of balloon surrounding a fist that has been pushed into it. The layer of balloon facing the
fist is the visceral layer; the outer layer is the parietal layer of the yolk sac. As the lumen of the yolk sac expands, the parietal layer
initially makes contact with chorion (the outermost embryonic membrane) which is in contact with the lining of the uterus. Shortly
thereafter, the yolk sac cavity deflates such that the distance between the visceral and parietal layers becomes quite small. Both the
chorion and the parietal layer of the yolk sac degenerate, resulting in the apposition of the visceral yolk sac wall to uterine luminal
lining. It is this organization (visceral yolk sac wall as the outer membrane) that leads to the description of this structure as an
inverted visceral yolk sac placenta. Nutrient-rich secretions from the glands of the uterine epithelium are transported across the
inverted visceral yolk sac placenta in a multistep process that includes being taken up by endocytosis or pinocytosis, digested by
lysosomes, and diffusion of the breakdown products across the epithelium (Freeman et al., 1981). This process is called histiotro-
phic nutrition. While this multistep transfer process is vulnerable to several sites of potential interruption, it is the major source of
nutrient supply to the short-gestation species prior to development of the chorioallantoic placenta. While humans also have a prom-
inent yolk sac during early gestation, the yolk sac does not expand as dramatically as in rodents and it never abuts the chorion/
uterine endometrium (Ramsey, 1982; Carney et al., 2004; DeSesso et al., 2012). In contrast to the inverted yolk sac placenta,
the late-developing chorioallantoic placenta establishes a cross- or countercurrent diffusion between maternal and fetal blood-
streams that efficiently transfers nutrients and gases.
The appearance of malformations after HBOC exposure primarily on GDs 8 and 9 coincides with the chronological appearance/
functioning of the inverted yolk sac placenta during GD 7; the rather abrupt disappearance of malformations in exposed litters after
GDs 10–11 corresponds well with the development of the chorioallantoic placenta of the rat, which becomes fully functional
during GD 11. Because the chorioallantoic placenta works by a different mechanism than the inverted yolk sac placenta and is
more efficient, the chorioallantoic placenta rapidly becomes the major physiological interface between the rat embryo and the preg-
nant dam. Thus, the data appear to be consistent with the idea that the early-developing inverted yolk sac placenta was the target
organ. Furthermore, reduction in nutrition to rat (Ellington, 1980) and mouse embryos (Szabo and Brent, 1975; Hemm et al.,
1977) during the early period of organogenesis has been linked to the production of malformations in these species, which is
also consistent with the findings in HBOC-treated litters.
The design of the studies that identified the inverted yolk sac placenta as the target organ involved careful controls to ensure that
the developmental toxicity was not due to increased oncotic pressure (HBOCs are colloids), or to increased maternal blood volumes
(dams were hemodiluted prior to infusion) (Stump et al., 2003, 2015). In addition, whole embryo culture experiments of rat
embryos in tissue culture media containing HBOC demonstrated that HBOCs inhibited the transfer of amino acids across the
visceral inverted yolk sac resulting in malformation and death of the exposed embryos (Stump et al., 2015).
The evidence for the absence of a likely impact of HBOC on human embryonic nutrition and gas exchange requires some further
discussion. Because the human yolk sac never abuts the chorion or uterine endometrium, transfer of substances from the maternal
system to the developing embryo via the yolk sac can only occur by means of diffusion into the celomic fluid that surrounds the
embryo with subsequent uptake from the yolk sac (a very inefficient mechanism). Some reports suggest that the human yolk sac
may absorb materials from the celomic fluid, however the small surface area of the human yolk sac indicates that histiotrophic
 Embryotoxicity: Anatomical, Physiological, Functional 29

nutrition via the yolk sac is not a major pathway for nutrient acquisition relative to the hemotrophic exchange occurring in the
chorioallantoic placenta (DeSesso et al., 2012). In contrast, the inverted visceral yolk sac placenta is the major site of maternal/fetal
exchange for several days. The role of the visceral yolk sac is important for the uptake of IgG and FcRn-containing biomolecules
(DeSesso et al., 2012; Bowman et al., 2013) in the uptake of toxicants has not been well-studied, although several molecules
(e.g., trypan blue, concanavalin A, HBOC) that inhibit yolk sac function cause developmental toxicity in rodents (Beck, 1976;
DeSesso et al., 1989; Holson et al., 2005). Consequently, developmental toxicity studies of HBOC were repeated using the dog:
a species that, similar to the human, never develops an inverted yolk sac placenta (Holson et al., 2015).
The dog was considered to be a relevant model not only because of its relatively long gestation period (Table 4) and the absence
of an inverted visceral yolk sac placenta, but also because HBOCs are approved for, and have been used safely in, veterinary treat-
ment of dogs. Groups of 20 pregnant Beagle dogs were infused with HBOC at doses of 6 g/kg on GDs 21, 25, 29, and 33 (Holson
et al., 2015). The dose is the maximum tolerated dose for canines over an 8 h period. The set of infusions results in an exposure that
is pharmacokinetically similar to that in the rat and provides exposure during the period of gestation that is equivalent to the sensi-
tive period in rats (Stump et al., 2003). At term, neither adverse effects on average fetal weights nor treatment-related malformations
after complete gross, visceral and skeletal examination were observed. These results, in concert with those of the rat developmental
toxicity studies and the rat whole embryo culture studies, highlight the absence of the target organ (the inverted yolk sac placenta) in
dogs as the reason that developmental toxicity did not occur.
Interruption of the processes of endocytosis, lysosomal degradation, and transport of digestant products across cells is expected
to transpire when absorptive epithelia are exposed to HBOCs. With respect for the ability of this physiologic action to be an under-
lying concern for developmental toxicity, it is noted that will only be a serious threat when these processes are the exclusive means
available to the embryo for obtaining. Because canine and human embryos never rely on histiotrophic nutrition as a sole means of
nourishment, interference with this physiologic process is irrelevant as a mode of action for developmental toxicity.

5.03.4 Case Studies: Functional Changes

The body of every animal functions, survives, thrives, and reproduces as a consequence of the complex interactions among its organ
systems. In mammals, these interacting organ systems begin to function more or less autonomously (e.g., without an overriding
maternal defense mechanism) after parturition. There are certain systems, such as the respiratory system, that do not function until
birth; other organ systems may malfunction in utero, but do not exert deleterious effects on the offspring until they must function
independently after birth (e.g., the urinary system); and still others are difficult to evaluate until they attain a level of capacity that is
developed after birth (e.g., the central nervous system). In these organ systems as examples, adverse health consequences of a dele-
terious exposure may not be manifested until the organ systems must operate on their own in the absence of a maternal safeguard.
Thus, it is quite possible that the precipitating event(s) could have occurred early in embryonic development and they could have
gone undetected until sometime after birth.
The set of case studies in this section describes endpoints that are observed after birth, each of which were elicited by exposure to
agents during gestation. The causes and delayed impacts discussed here include faulty functioning of the lung (respiratory distress
syndrome caused by cadmium), urinary system (altered kidney function due to prenatal exposure to fluorine or mercury), and the
central nervous system (putative causes of autism and autism spectrum disorder).

5.03.4.1 Cadmium and the Developing Lung

The lung develops as a ventral, midline out pocketing of the foregut, the respiratory diverticulum, which appears during the 5th
week of gestation in humans. Its embryology is described in numerous embryology texts (e.g., Carlson, 2014; Sadler, 2006;
Schoenwolf et al., 2008) and will be reviewed quickly here. The respiratory diverticulum is composed of endoderm surrounded
by splanchnic mesenchyme. It grows quickly, and soon bifurcates into two laterally positioned lung buds. With continued growth,
the lung buds each subdivide such that the left bud has two major divisions (corresponding to the two lobes of the left lung)
whereas the right lung bud has three (corresponding to the three lobes of that lung). The repeated divisions of the duct system
(that will conduct air into the presumptive lung) continue throughout gestation such that in humans, there are 23 bifurcations prior
to birth (Thurlbeck, 1975; Burri, 1974; Burri et al., 1973). The maturation of the lung occurs in stages, which are described by the
morphological characteristics of the developing airway system (Boyden, 1977; Zeltner and Burri, 1987). Because of the ongoing
branching of the duct system of the lungs, the duration of the stages overlap within each species and due to differences in the lengths
of gestation, the overall state of maturation of the lung differs from species to species (Zoetis and Hurtt, 2003a). For instance, at
birth in humans only approximately 1/8–1/6 of the adult number of alveoli are present. The majority of the adult complement
of alveoli is formed by 2 years of age, although alveoli are likely still being formed at a low rate until age seven. In contrast, rats
have no alveoli at birth; alveoli appear at postnatal day 4 and alveolar development is essential complete by 2 weeks of age (Zoetis
and Hurtt, 2003a). A critical phenomenon for efficient lung function is the appearance of type II alveolar cells during the final stage
of lung maturation and the secretion of pulmonary surfactant, which reduces surface tension and thereby enhances the expansion of
the alveoli. Absence/reduction of pulmonary surfactant hinders the performance of the lung in the newborn, resulting in respiratory
distress. In the following case study, in utero exposure to cadmium during organogenesis caused changes in lung function that are
consistent with respiratory distress.
30 Embryotoxicity: Anatomical, Physiological, Functional

Daily subcutaneous injections of cadmium chloride (8 mg/kg) on GDs 12–15 into pregnant rats caused delayed parturition,
reduced mean fetal body weights, and respiratory distress syndrome (but no malformed lungs) in 11% of offspring (Daston and
Grabowski, 1979). At necropsy, the lungs of the pups that showed signs of respiratory distress were a deep purple (compared to
the pink color expected) and histological examination of revealed collapse of many alveoli with refractile eosinophilic membranes
(hyaline membranes) lining the airway surface. The affected lungs were found to have a decline in the absolute amount of lecithin,
an essential component of surfactant (Daston and Grabowski, 1979); reduced glycogen content compared to controls (Daston,
1982); and to be deficient in the rate of incorporation of choline into phosphatidylcholine (another important component of
surfactant) during the last days of gestation (Daston, 1982).

5.03.4.2 Mercury and Fluorine and the Developing Kidney

The definitive kidney develops in the pelvic area from the uteric bud, an evagination of the mesonephric (Wolffian) duct, and a mass
of intermediate mesoderm cells, the metanephric blastema. The uteric bud contacts the metanephric blastema, invades the tissue
mass, and branches repeatedly to form the collecting system; at the distal regions of the uteric bud, interactions with the mesen-
chyme of the metanephric blastema induce the functional units of the kidney: the nephrons. The earliest nephrons are formed
in what will be the more centrally located portion of the kidney; the cortical region will be the last region to complete nephrogen-
esis. The sequence of events in various vertebrate species is similar, although the timing differs, especially with regard to those events
completed prior to birth in shorter gestation species (Zoetis and Hurtt, 2003b; Evan et al., 1984). For instance, formation of neph-
rons is completed in humans by the 35th week of gestation whereas in rats, this process is not complete until 4–6 weeks of postnatal
life (Baxter and Yoffey, 1948; Zoetis and Hurtt, 2003b).
Daily prenatal injection of methylmercury (0.5 of 1.0 mg/kg sc) into pregnant rats beginning on GD 8 and continuing until term
caused no gross malformations or other alterations observable at birth in the offspring, although postnatal growth exhibited a small
decrease relative to controls and an increase in kidney to body weight ratio by 33 days of age (Slotkin et al., 1986). Despite the
absence of overt developmental toxicity, renal function in the treated offspring was impaired as evidenced at 3 weeks of age by
decreased creatinine clearance, increased fractional excretion of sodium, and (in the low dose group only) increased fractional excre-
tion of urea and osmotic particles. The alterations seen after prenatal exposure are as dramatic as those seen when pups are dosed
directly, but this may be related to the lower exposures of the offspring when mediated via placental transport.
It is of note that sodium fluoride and mercuric chloride, both potent nephrotoxicants in the adult rat, cause little effect on the
still-developing neonatal kidney (Daston et al., 1983, 1985) and that sensitivity to these compounds increases after weaning, as the
kidney matures. These latter data demonstrate that young and developing animals are not always more susceptible than adults to
the effects of toxicants. Further, they demonstrate that it is important to ensure that the animal models used to assess risks for age-
specific toxicity are at the same developmental stage as the humans who are to be protected (Morford et al., 2004).

5.03.4.3 Autism and Autism Spectrum Disorder

The early development of the nervous system was mentioned in the inorganic arsenic case study. As with all organ systems, nervous
system development is not complete at birth and continues during postnatal life. The extent of postnatal development depends not
only on the length of the gestational period for each species, but also on the complexity of the brain. Watson et al. (2006) prepared
a comparative morphological assessment of postnatal brain maturation in several species. Maturation of the nervous system during
postnatal life is associated with behaviors that emerge as children become increasingly more interested in social interactions and
communication. Alterations in these behaviors are seen children who exhibit autism and autism spectrum disorders. Exposure
to several human teratogens during the first trimester has been associated with autism or autism spectrum disorder (Arndt et al.,
2005). The strongest associations were for maternal infection with rubella and exposures to thalidomide, valproic acid, and miso-
prostol. In some of these cases, there were unrelated morphological observations such as slight changes in facies or genitalia. Of
interest here is that Rodier et al. (1997) reported that some populations of children with idiopathic autism shared some of these
dysmorphic features.
With respect to the developing central nervous system, gross malformations of the brain are usually not apparent. At autopsy,
a variety of occasional and hard-to-see anatomic changes have been reported, including alterations in the pyramidal decussation
(Bailey et al., 1998), dysplasia of the cerebellar vermis, and the facial colliculus (due to reduced numbers of motor neurons in
the facial nucleus; Rodier et al., 1996). Alterations are usually much less apparent and have been found only after histological exam-
ination of cerebellum and brainstem, which has revealed reductions in the numbers of neurons expected in a variety of structures
neurochemical analyses of monoamine concentrations, which revealed increases in serotonin and dopamine (discussed in Arndt
et al., 2005). The details surrounding the potential for evelopmental exposures to contribute to autism or autism spectrum disorder
are not so well established as those of the other cases studies. Nevertheless, while the cause(s) and natural history of autism are in
the early stages of being elucidated, it is important to note that what may appear as only a subtle change in the nervous system due
to an exposure during early gestation can have a dramatic effect on a child’s cognitive behavior during postnatal life.
Taken together this set of case studies demonstrates that, even in the absence of grossly observable malformations, develop-
mental toxicity can be manifested by impaired function that can greatly diminish the quality of life of the affected individual
and his or her loved ones.
 Embryotoxicity: Anatomical, Physiological, Functional 31

5.03.5 Conclusion

The development of an embryo is an extraordinarily complex, multifaceted process that occurs under severe constraints of schedules
for gene expression/repression, the opportune delivery and acquisition of nutrients to enable synthesis of molecular signals as well as
structural molecules, and timely mitosis or apoptosis. It comes as no surprise that disturbance of the process can impact the quality
of the end product. Through a series of case studies, this chapter has attempted to provide insight as to how the various levels of
organization within the embryo can be affected and contribute to impaired development. The examples included grossly observable
malformations, as caused by injection of inorganic arsenic into rodents during the sensitive period; retarded growth and maldevel-
opment when the physiology of the inverted yolk sac placenta is compromised at a critical time in development; and diminished
function of organ systems such as the kidneys, lungs, and central nervous system after exposure during early embryogenesis.
Embedded within this set of examples, the chapter touched on concepts of critical periods of development (when the embryo is
most sensitive to perturbation by xenobiotics); the way the maternal organism (and her pharmacokinetics) affects her offspring;
the need for and role of placentae in nutrition and toxicity; and the need for threshold concentration exceedance to elicit embryotox-
icity. The chapter ended with several cases in which the typical, overt signs of developmental toxicity were not present, although the
effects of adverse exposure during embryonic life are manifest throughout a life that will be reduced in quality and shortened in time.

See also: 5.11. Altered Gene Expression in Diabetic Embryopathy: Multiple Pathways in Analysis and Interpretation. 8.29. Physiological
and Pathological Functions of Mammalian MicroRNAs. 15.10. Environmental Exposures and Developmental Programming of the
Lung.

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Further Reading

Tabocova, S., Hunter, E. S., & Gladen, B. C. (1996). Developmental toxicity of inorganic arsenic in whole embryo culture: Oxidation state, dose, time, and gestational age
dependence. Toxicology and Applied Pharmacology, 138, 298–307.