Maternally Mediated Developmental Toxicity☆

5.06

Maternally Mediated Developmental Toxicityq

C Harris, University of Michigan, Ann Arbor, MI, United States JM Rogers, U.S. Environmental Protection Agency, Research Triangle Park, NC, United States © 2018 Elsevier Ltd. All rights reserved.

5.06.1 5.06.2 5.06.2.1 5.06.2.2 5.06.3 5.06.3.1 5.06.3.2 5.06.3.2.1 5.06.3.2.2 5.06.3.2.3 5.06.3.3 5.06.3.4 5.06.3.5 5.06.4 5.06.5 References Further Reading

Introduction Impact of General Maternal Effects on Prenatal Development Lower Maternal Weight Gain and Food Intake, and Malnutrition Maternal Stress Specific Perturbations of Maternal Physiology: Impact on Prenatal Development Acid–Base Imbalance Micronutrient Deficiencies Zinc deficiency Folic acid deficiency Choline deficiency Maternal Cardiovascular Disturbances Hyper- and Hypoosmolality Altered Body Temperature Data Evaluation: Unraveling Maternal and Developmental Toxicity Findings New Concepts in Maternal Toxicity for Safety Evaluation

86 87 87 89 90 90 91 92 93 93 93 94 94 95 97 97 99

Abbreviations AMP Amino-2-methylpropanol BW Body weight CA Carbonic anhydrase CCM2 Cerebral cavernous malformation 2 DOHaD Developmental origins of health and disease EDTA Ethylenediaminetetraacetic acid EG Ethylene glycol GD Gestation day Hgb Hemoglobin IUGR Intrauterine growth retardation LPD Low protein diet MAPK Mitogen-activated protein kinase MT Metallothionein RBC Red blood cell

5.06.1

Introduction

According to Karnofsky’s law (Karnofsky, 1965), all chemical agents, whether natural or man-made, have the potential to adversely affect in utero development provided that exposure occurs at a sufficiently high dose, administered by the correct route, at the right time in pregnancy. How is it then that society can tolerate the use of countless different chemical agents and feel reasonably assured

q

Change History: April 2017. JM Rogers and C Harris made updates throughout the chapter, adding more recent references where appropriate. The authors expanded the treatment of maternal nutrition, including effects of low protein diet and deficiencies of micronutrients. They expanded the discussion of the long-term effects of the developmental environment, including the Developmental Origins of Health and Disease (DOHaD) theory. The section on unraveling the contribution of maternal toxicity to the developmental toxicity of AMP (Ball et al, 2014) has been added. A short section on maternal microflora and the role of the microflora in development has also been added. The section on histiotrophic nutrition has been expanded and updated. This is an update of EW Carney, Maternally Mediated Developmental Toxicity, Comprehensive Toxicology, Second Edition, edited by Charlene A. McQueen, Elsevier, Oxford, 2010, Volume 12, Pages 163–176, ISBN 978-0-08-046884-6, 10.1016/B978-0-08-046884-6.01516-5.

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that these will not harm our future generations? The answer, of course, is that scientists and public health policy makers have devised a safety assessment system in which pharmaceuticals, pesticides, agrichemicals, industrial chemicals, cosmetics, and other regulated agents are evaluated using standardized tests in pregnant laboratory animals. Specific guidelines for the conduct of these studies have been in place for several decades, and although they have been refined over the years, they have always required administration of test chemicals to pregnant animals, most commonly rats and rabbits, followed by assessment of the full-term fetuses for any untoward effects on viability, growth or morphology. To maximize the detection of effects, these test guidelines require that a range of dosages be administered, including a high dose level that causes overt toxicity in the pregnant animal. Given the dependence of the mammalian embryo/fetus upon the maternal system, it should come as no surprise that maternal toxicity may have adverse effects on developing offspring. An extensive review of 234 chemicals tested for developmental toxicity showed that 75% of them showed developmental effects only at doses that also caused maternal toxicity (Khera, 1985). What are the implications for this three-fourths majority of chemicals that show developmental toxicity only at maternotoxic dose levels? The evolution of thought on this issue has been documented in a number of workshops and publications (Beyer et al., 2011; Chahoud et al., 1999; Chernoff et al., 2008, 1989; DeSesso, 1987; Keen, 1992; Khera, 1987; Palmer and Kavlock, 1987; Skalko and Johnson, 1987). For many years, developmental toxicity that was concomitant with maternal toxicity was often dismissed as a secondary effect and had little impact on chemical regulation. Others have argued that it does not matter whether developmental toxicity is secondary to maternal toxicity or a primary effect on the embryo, as risk assessments are simply based on the most sensitive effect in the study, regardless of whether that effect is secondary to maternal toxicity or not. In between these two extremes is the reality that maternal toxicity is an umbrella term for a constellation of possible maternal physiological perturbations, and the extent to which any one of them adversely affects development depends on the target organs involved, severity of the toxicity, and the stage of gestation at which it occurs. Studies designed to explore the relationship of maternal toxicity with specific adverse developmental outcomes have demonstrated that the relationship is complex. Kavlock et al. (1985) dosed CD-1 mice on day 8 of gestation with one of ten chemicals at dosages predicted to produce a low to moderate incidence of maternal mortality. Effects on pregnancy outcome included complete litter loss (three chemicals), reduced fetal viability (one chemical), lower fetal weight (three chemicals), and terata (two chemicals). The most consistent finding across these chemicals was an increased incidence of supernumerary lumbar ribs (seven of ten chemicals), and there was an inverse relationship between maternal weight gain during gestation and the incidence of supernumerary ribs in the treated groups. Chernoff et al. (1990) carried out similar studies in Sprague-Dawley rats, dosing dams with one of five different chemicals on gestation days 6–15 at dosages causing maternal mortality or lower weight gain during pregnancy. Decreased fetal weight (two chemicals) and viability (two chemicals), supernumerary ribs (two chemicals) and enlarged renal pelves (two chemicals) were the treatment-related effects observed. Thus, there was little consistency in the profile of adverse pregnancy outcomes in the face of significant maternal toxicity during pregnancy. Understanding the mode of action for a given chemical and particularly, whether or not that mode of action is maternally mediated, can greatly increase the level of confidence in extrapolation of the animal data to human risk assessment. For example, if maternal toxicity is found to be a required event in the mode of action, one can then ask whether or not this event is relevant to humans under various real-world exposure scenarios (Seed et al., 2005). If not, concerns for developmental toxicity in humans should be much less than if maternal toxicity is a realistic possibility under typical human exposure scenarios. The outcome of such evaluations can have a profound impact on regulation, particularly with respect to chemical classification and labeling. Given the impact of maternal toxicity on chemical regulation, there is renewed interest in the relationship between maternal toxicity and developmental toxicity. Toward this end, this chapter reviews mode of action studies that have explored the relationship between specific manifestations of maternal toxicity and their resulting impact on the developing embryo or fetus. Departing from past approaches that have tended to be more phenomenological in nature, the approach taken here is to delve into the physiology of maternal toxicity by characterizing the key stressors presented to the conceptus, and then assessing the conceptus’s ability to cope with such changes. Where possible, an attempt will be made to delineate the magnitude of the maternal effect, as well as the time during pregnancy at which it adversely affects development. The chapter will close with some guidance on interpreting data relevant to the maternal toxicity–developmental toxicity relationship, and an outline of newer approaches for dealing with this issue.

5.06.2

Impact of General Maternal Effects on Prenatal Development

5.06.2.1

Lower Maternal Weight Gain and Food Intake, and Malnutrition

In prenatal developmental toxicity safety assessment studies, a significant reduction in maternal body weight gain is the typical way in which the required evidence of maternal toxicity at the highest dose level is demonstrated. Changes in maternal weight gain could be brought about by specific target organ toxicity, or something as mundane as reduced food intake triggered by the taste or smell of the compound. To determine the impact of reduced maternal body weight on specific developmental toxicity endpoints, a number of animal studies have employed various food restriction regimens during pregnancy in order to decrease maternal weight gain, and then have assessed the impact of these regimens on the full-term fetus. In both rabbits and rats, it appears that the most sensitive and consistent fetal response to maternal food restriction and/or lower body weight gain is fetal growth retardation, often accompanied by reduced skeletal ossification, particularly in the calvaria, sternebrae, thoracic centra, and phalanges (Carney and Kimmel, 2007). In the most recent and detailed of the rabbit studies listed in

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Table 1

Food restriction studies in pregnant rabbits and rats

Food restriction regimen Gram per day Gestation days

Maternal effects

Body weight gain a Abortions (%) Y Fetal BW Fetal death Y Ossification Malformations References

Rabbit 110 75 55 35 15 150 75 15 50 15 150 60 20

6–18 (ad lib 19–30) 6–18 (ad lib 19–28) 6–20 (ad lib 21–29)

Rat 20 15 10 7.5

6–17 Y (ad lib GD 18–21) Y BW loss BW loss

7–19 (ad lib 20–29)

Developmental effects

Y Y Y BW loss Y BW loss BW loss BW loss BW loss BW loss BW loss

0 0 7 0 40 0 0 17 7 12 0 0 86

Y 3% Y 5%b Y 10%b Y 14%b Y 16%b [ 3–6% Y 3% Y 9%b Y 5% Y 13%b [ 8% Y 5% Y 37%b

0 0 0 0

Y 5%b Y 7%b Y 10%b Y 24%b

Cappon et al. (2005) O O O O Petrere et al. (1993)

O O

NA

Clark et al. (1986) O NA NA NA

O Matsuzawa et al. (1981)

Fleeman et al. (2005)

O

a

Weight gain during treatment. In all studies, body weight gains were increased in the posttreatment recovery period. Significantly different from control. GD, gestation day; NA, not applicable. Only one litter in the 20 g day
1 group; skeletal examinations not conducted in any group. b

Table 1, fetal body weights were significantly decreased when mean maternal body weight gains during the treatment period were reduced by 24% (Cappon et al., 2005). It is important to realize that these studies restricted food only until the end of the embryonic period so as to mimic the standard dosing period for pharmaceutical safety studies. The guidelines for assessment of pesticides and environmental chemicals require dosing until full term. Given that the demands of fetal growth become extremely intense during the last few days of gestation, reductions in maternal food intake and/or body weight gain that continue into late gestation would undoubtedly result in even greater effects on fetal body weight and skeletal ossification. In an analysis of 125 National Toxicology Program developmental toxicity studies in the mouse, rat and rabbit, Chernoff et al. (2008) found that lower fetal weight was often associated with lower maternal weight gain late in pregnancy, and that the degree of fetal weight reduction was correlated with the extent of the decrement in maternal weight gain. Another consistent response to severely reduced food intake is spontaneous abortion in rabbits (Cappon et al., 2005; Matsuzawa et al., 1981; Petrere et al., 1993). Rabbits can be particularly prone to gastrointestinal upset or alterations in their gut flora (Clark et al., 1986), with individual rabbits going completely off food for days at a time for no apparent reason. When this period of severe inanition occurs in mid- to late-pregnancy (e.g., gestation day (GD) 19-term), abortions are likely, especially if the doe experiences body weight loss (Cappon et al., 2005). These food consumption- and body weight-related abortions generally do not occur in rats. In contrast to the effects of reduced maternal food intake on fetal growth and skeletal ossification, the overall weight of evidence from these and other food restriction studies indicates that even severe inanition or body weight effects do not alone result in embryo/fetal lethality or structural teratogenesis. The most extreme cases of reduced nutrient intake during pregnancy are those cases presenting with varying degrees of maternal malnutrition ranging from selective micronutrient deficiencies to outright famine. The overall causes of reduced nutrient intake cover a broad spectrum of physiological, environmental and disease factors. In addition to a large body of literature on experimental animal studies (see Armitage et al., 2004 for review), a growing body of literature from human studies is also now available, describing the effects of human malnutrition and nutrient deficits on embryo/fetal outcomes as well as the postnatal effects across the life course. Maternal undernutrition, in the form of low protein diets (LPD) in experimental animals, has been demonstrated to elicit biochemical, molecular, and morphological abnormalities in developing prenatal offspring. The most consistently affected tissues appear to be those with prominent brush borders and high active transport capacity such as the trophoblast cells of the rat blastocyst (Gao et al., 2013), visceral yolk sac, and the kidney proximal tubules (Cornock et al., 2010; Harrison and Langley-Evans, 2009). In hens maintained on low protein diets, yolk and visceral yolk sac membranes in embryos showed significant changes in their expression of leptin and glucocorticoid receptors believed to be related to observed growth deficits (Rao et al., 2009). In rodents, maternal LPDs were found to impair nephrogenesis and promote hypertension (Langley-Evans et al., 1999), up-regulate cholesterol and lipoprotein transport, atherosclerosis signaling, clathrin-mediated endocytosis and LXR/RXR and FXR/RXR activation. Many of these studies focused on later stages of development where the placenta was the main target for selective changes in expression

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and function elicited by the maternal LPD. Deleterious consequences appeared in the offspring as secondary outcomes. In conjunction with these changes, gene expression for multiple apolipoproteins, triglyceride transfer proteins, retinol and thyroid hormone transporters and cubilin, the receptor-mediated endocytosis receptor protein, were all increased several fold. There is a paucity of information available to assess whether maternal LPDs can also directly elicit changes in gene expression, nutrient protein uptake, or metabolism in the visceral yolk sac placenta at earlier stages of embryogenesis. Several well-documented occurrences of human malnutrition and consequences of prenatal famine have been extensively studied, including multiple occurrences caused by crop failure and seasonal famines (Sweden, Finland, Gambia, and Bangladesh), as well as famines resulting from war and political oppression (Dutch Hunger Winter of 1944–45, Siege of Leningrad 1941–44, Chinese Great Leap Forward 1959–61) (Lumey et al., 2011). The first observed consequences of maternal malnutrition/famine during gestation are low birth weights. Overt anatomical and functional malformations are not widely reported in malnourished humans except in cases of specific micronutrient deficiency, such as is reported for folic acid status and the incidence of neural tube defects (McDonald et al., 2003; Czeizel, 2009). Recent studies have, however, suggested that the suboptimal maternal nutrition that accompanies exposure to chemical agents such as the teratogen ethanol may also be an important contributor to their teratogenic action (Keen et al., 2010; Young et al., 2014). Many of the consequences of early developmental in utero nutritional deficiency are now believed to significantly impact the critical epigenetic programming of the embryo/fetus which can lead to altered metabolism and increased sensitivity to various diseases later in postnatal and adult life. This epigenetic reprogramming process forms the basis of the “developmental origins of health and disease” (DOHaD) research paradigm that has been extensively reviewed elsewhere (Barker et al., 2006; Godfrey and Barker, 2000; Lau et al., 2011; Seckl, 2004) and is a topic of active ongoing investigation. The early life nutritional conditions that lead to postnatal and adult susceptibilities to disease and metabolic disorders are dependent on the timing and stage of prenatal development, and early stages of development show the greatest vulnerability. Increased risk of schizophrenia, greater responsiveness to stress, increased atherogenic plasma lipid profiles, doubled rates of coronary heart disease, premature loss of cognitive ability, increased type 2 diabetes, metabolic syndrome, obesity, IUGR, and many other outcomes (Langley-Evans, 2015; Bhasin et al., 2009; Bonora et al., 2003; Lumey et al., 2011) are known to occur at higher frequency following maternal nutritional deficits during pregnancy. Further examples of specific maternal micronutrient deficiencies will be discussed below. Alterations in human maternal microflora in the gut, vagina, placenta and oral cavity are also drawing increased attention for their ability to influence a number of developmental processes including growth, morphogenesis, and proper programming of the immune system (Nuriel-Ohayon et al., 2016). The general composition of the maternal microbiome changes naturally throughout gestation to meet the metabolic needs of the mother as well as the changing developmental needs of the embryo and fetus. Several discrete developmental events that are directly regulated by maternal hormones have also been shown to be impacted by the hormone metabolizing activities of microbiota. Deviations from normal embryonic and fetal development can be elicited indirectly through environmental factors affecting the maternal microflora, such as the maternal diet, disease, and the use or misuse of antibiotics. In principle, the maternal microbiome serves as the seed source for the fetal microbiome. The types and quantities of microbes passed from the mother can play an important role in the occurrence of adverse developmental outcomes because of changes in growth factors and nutrients passed from the maternal side to the conceptus with resultant changes in embryo/fetal metabolism (Nuriel-Ohayon et al., 2016).

5.06.2.2

Maternal Stress

From an anthropomorphic perspective, experiencing chemically induced maternal toxicity is presumed to be inherently stressful. As such, many have hypothesized that maternal stress could contribute to the developmental toxicity that often accompanies maternal toxicity. To study this question, researchers have shown remarkable creativity in devising various models of stress induced through the use of flashing lights, alarms, buzzers, jet engine noise, forced exercise, restraint, mild shocks, and the smell of urine from predator animal species. It is difficult to know if such models, which probably induce anxiety, adequately mimic the stress response of a laboratory animal experiencing chemically induced toxicity. Indeed, the concept of generalized or universal stress is an oversimplification. Different stressors clearly can activate widely different neuronal pathways (Harbuz and Lightman, 1989; Szekely, 1990) and elaborate diverse combinations of endogenous opioids, hypothalamic releasing factors, and cytokines (Szekely, 1990). Another caution with these models of stress is that the levels of corticosterone and other stress hormones could be much higher than those that occur in scenarios of chemically-induced maternal toxicity. Also, physical changes such as increased abdominal pressure during prolonged restraint or hyperthermia during forced exercise are potential confounders that may produce artifactual effects on prenatal development. Keeping the aforementioned caveats in mind, a number of these studies have reported effects that have included decreased litter size and birth weight, embryo mortality, and/or abortion (Euker and Riegle, 1973; Geber and Anderson, 1967; Geber et al., 1966; Kholkute and Udupa, 1978; Matsuzawa et al., 1981), although other studies using similar procedures failed to induce adverse effects on development (Nawrot et al., 1980; Tyl et al., 1994). Restraint stress, in which pregnant animals have been physically immobilized for up to 12 h at a time on different days of gestation, has caused implantation failure, supernumerary ribs, and cleft palate, with mice being more susceptible than rats (Beyer and Chernoff, 1986; Chernoff et al., 1987, 1990). Administration of glucocorticoids (e.g., dexamethasone) during pregnancy is known to cause developmental toxicity, but whether or not endogenous levels of glucocorticoids associated with maternal toxicity are elevated to a sufficient extent to affect development is debatable (Barlow et al., 1980; Daston, 1994; Eldeib and Reddy, 1990; Hansen et al., 1988).

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While the contribution of maternal stress to standard prenatal toxicity endpoints remains uncertain, there is strong evidence that stress during in utero development can affect postnatal development. Reported effects in rodents include demasculinization of sexual differentiation (vom Saal et al., 1990; Ward and Weisz, 1984), increased anxiety, and altered mating behavior, estrous cyclicity, learning, memory, and ability to adapt to stressful stimuli (Baker et al., 2008; Lau et al., 1996), and alterations in the programming of the hypothalamic-pituitary- adrenal axis (reviewed in Weinstock, 2008 and Darnaudéry and Maccari, 2008).

5.06.3

Specific Perturbations of Maternal Physiology: Impact on Prenatal Development

5.06.3.1

Acid–Base Imbalance

Among the most fundamental of maternal physiological functions is maintenance of extracellular fluid pH, which is essential for the normal function of enzymatic and numerous other pH-dependent processes in the body. Blood pH is controlled within extremely narrow limits (7.36–7.44 in humans), which is a good thing, as a drop of just 0.5 pH unit can be fatal. To avoid such lethal excursions, the maternal system is armed with several different buffer systems, among them bicarbonate/carbonic acid/CO2, plasma proteins, plasma phosphate, and hemoglobin (Fig. 1A). However, the capacity of these systems to maintain pH is finite, and extracellular fluid pH can become excessively basic or acidic following exposure to bases or acids, in response to toxic injury of the lungs or kidneys, or in conjunction with several disease states, such as diabetes mellitus (Sestoft and Bartels, 1983). When faced with an acid load (acidosis), the immediate compensatory strategy is to neutralize with endogenous base, resulting in a drop in blood bicarbonate (HCO
3 ) concentration. The respiratory centers respond quickly by increasing pulmonary ventilation rates, with blood pCO2 quickly falling to maintain the normal ratio of CO2 to HCO
3 (Blechner, 1993). Reductions in pCO2 and
HCO
3 may last for several hours or more as restoration of normal levels is a slower process limited by the rate of HCO3 reabsorption in the kidney (Sestoft and Bartels, 1983). Respiratory acidosis occurs when pulmonary ventilation is inhibited, and is characterized by increased pCO2 and increased renal HCO
3 reabsorption. Acetozolamide causes a mixed respiratory/metabolic acidosis stemming from its inhibitory action on carbonic anhydrase, the enzyme that catalyzes the interconversion of CO2, carbonic
acid, and HCO
3 . In this case, pCO2 is increased, HCO3 increases slightly, and pH decreases (Weaver and Scott, 1984). Metabolic and respiratory alkaloses also have their own characteristic profiles (Blechner, 1993; Sestoft and Bartels, 1983). The effect of maternal acid–base imbalances on embryo development was examined in a landmark study by Khera (1991), who dosed pregnant rats with high doses of ethylene glycol (EG) or sodium salicylate, either given alone or in conjunction with a sodium bicarbonate infusion to maintain normal acid–base balance. With each chemical, prevention of metabolic acidosis via bicarbonate infusion resulted in a significant reduction in developmental toxicity relative to that caused by EG or salicylate alone, although the reduction in developmental effects was by no means complete. Subsequent studies on EG sought to further delineate the role of acidosis in developmental toxicity by comparing the developmental effects of EG’s metabolite, glycolic acid, to an equimolar dose of sodium glycolate solution at pH 7.4 (Carney et al., 1999). Maternal blood levels of total glycolate (free acid and glycolate anion combined) were confirmed to be essentially identical; however, glycolic acid induced metabolic acidosis whereas sodium

Fig. 1 Overview of acid–base regulation in (A) maternal blood and other extracellular fluids, and (B) the intracellular environment of the embryo (rat pH values used as an example). CA, carbonic anhydrase.

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glycolate had no impact on acid–base parameters. Sodium glycolate treatment caused increases in skeletal malformations and variations, as well as a decrease in fetal body weight, demonstrating that metabolic acidosis was not required for developmental toxicity. On the other hand, the incidence of developmental effects was considerably higher in the group given glycolic acid, suggesting that metabolic acidosis was either causing some developmental effects directly or that it was somehow interacting with glycolate to exacerbate toxicity. To address this question further, we must consider that acid–base disturbances in vivo not only involve changes in pH, but CO2 and bicarbonate as well. In vitro studies on both pre- and postimplantation embryos have revealed a surprising ability to develop normally at pHs ranging from approximately 6.5 to 8.0 (Andrews et al., 1995; Carney and Bavister, 1987; Carney et al., 1996; Kane,
1987). Data on HCO
3 are limited, but indicate that excess HCO3 is not developmentally toxic (Goldman and Yakovac, 1964;
Khera, 1991). The lack of direct effects of pH or HCO3 is consistent with the embryo’s expression of HCO
3 /chloride exchangers and Naþ/Hþ antiporters that are effective at regulating intracellular proton and HCO
3 levels (Steeves et al., 2001) (Fig. 1B). Interestingly, the pH of embryonic cells and extraembryonic fluid compartments appears to be regulated independent of plasma pH, and also changes in a stage-specific manner. For example, the intracellular pH of mouse embryos was 7.65, 7.64, and 7.41 on GD 8, 9, and 10, respectively, despite the fact that maternal plasma pH remained stable at pH 7.26  0.05 (Bell et al., 1997; Scott and Nau, 1987). In monkeys, the intracellular pH of embryo homogenates changed from 7.35 on GD 24–29 to 7.15 on GD 30–31 and to 6.74 on GD 36–37, while maternal blood pH remained stable at 7.33–7.35 (Collins et al., 1996). All of this suggests that embryos are able to withstand fairly large changes in proton and bicarbonate concentrations in the maternal environment. The impact of altered CO2 concentrations may be somewhat different, as there is evidence indicating that very high atmospheric concentrations of CO2 (hypercapnia) are teratogenic in rats (Haring, 1960), rabbits (Grote, 1965), and certain strains of mice (Weaver and Scott, 1984). Also, cells of the embryo do not have an effective means of keeping CO2 out, as CO2 freely crosses cell membranes. The proposed mechanism by which CO2, as well as carbonic anhydrase inhibitors (e.g., acetozolamide), cause teratogenesis involves their ready transfer into embryonic cells, which then leads to acidification of intracellular pH (Bell et al., 1997; Scott and Nau, 1987). Intracellular pH is a critical regulator of many developmental processes, and changes as small as 0.1–0.2 units can significantly alter development (Busa and Nuccitelli, 1984). While the direct effects of acid–base disturbances remain uncertain, an even more important role for maternal acid–base disruption is its potential influence on placental transfer of toxicants. As mentioned, there are gradients between the pH of maternal blood and various conceptus compartments that have been shown to affect the transplacental distribution of weak acids and bases. For example, weak acids tend to concentrate in the GD 8 mouse embryo because it is more alkaline than maternal blood (Nau and Scott, 1986). A maternal metabolic acidosis would increase the size of this pH gradient and result in an even higher dose of toxicant to the embryo. Indirect evidence supporting this hypothesis comes from an embryo culture study in which acidic pH induced a leftward shift in the dose–response curve of sodium formate (Andrews et al., 1995). A study using the drugs bupivacaine and meperidine also indicated that acidosis may affect placental transfer, as more drug was delivered to rabbit fetuses when the perfusate was adjusted to pH 7.0 as compared with pH 7.5 (Gaylard et al., 1990).

5.06.3.2

Micronutrient Deficiencies

Chemical exposure may result in maternal alterations in micronutrient levels either due to the intended action of the drug or chemical or as an indirect consequence associated with tissue damage and induction of an acute phase response. These maternal alterations may stem from inhibited gastrointestinal tract absorption, sequestration and enhanced elimination (e.g., chelation), and/or redistribution from one organ to another, which would include altered placental transfer. How a given micronutrient is stored within the maternal body and the kinetics of its depletion will have a large bearing on the impact on development. For some micronutrients (e.g., retinol), hepatic storage pools act as a capacitor buffering against short-term changes in plasma levels, whereas mobilization of zinc stores is limited during periods of decreased supply, such that short-term zinc deficiency can rapidly affect maternal blood and embryonic levels of zinc (Ashworth and Antipatis, 2001). The pathways and methods for maternal transfer of micronutrients to the developing embryo/fetus differ significantly as a function of developmental stage and can vary across species. Following fertilization and prior to implantation, the embryo acquires its nutrients from oviduct fluid, which is composed of a selective but complex maternal plasma transudate along with the secretion of some proteins formed in the oviduct epithelium. Nutrients, including pyruvate, lactic acid and glucose are selectively taken up and utilized by the embryo to support its growth needs. Studies in rodents show that maternal undernutrition during the preimplantation period, caused by a low protein diet (LPD) produced low cell numbers in the inner cell mass and malformations of the blastocyst. The effects on the blastocyst were concomitant with reduced levels of insulin, essential amino acids, and glucose in maternal serum (Kwong et al., 2000). Following implantation, the nutritional relationship changes as the conceptus is temporarily removed from direct contact with maternal serum as a result of interstitial implantation. During this phase, which encompasses embryonic organogenesis, the conceptus relies on the process of histiotrophic nutrition to capture and process nutrients to meet its growth and metabolic needs. The near exclusive reliance on histiotrophic nutrition in rodents and rabbits during postimplantation embryogenesis suggests that conceptal availability of maternal proteins may be of greater importance for embryonic nutrition than the quality and quantity of micronutrients supplied via the maternal circulation prior to placentation (Marshall et al., 2015). Histiotrophic nutrition consists of receptor mediated endocytosis of whole bulk proteins and specific carrier proteins along with their respective cargoes followed by accumulation into acidified endocytotic vacuoles. Cargo molecules such as vitamins, cofactors,

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Maternally Mediated Developmental Toxicity

growth factors and minerals are captured by the brush border endoderm of the visceral yolk sac and the remaining proteins are proteolytically degraded in lysosomes. Evidence from recent studies show that first trimester human visceral yolk sacs capture and utilize endometrial gland proteins as a nutrient source, suggesting that maternal protein malnutrition and reduced endometrial gland secretion could have a significant negative impact on the growing embryo (Burton et al., 2001, 2002). The critical role of histiotrophic nutrition during organogenesis has been well characterized in rodents and rabbits but there is still considerable debate as to its role in human development (Holson et al., 2005).

5.06.3.2.1

Zinc deficiency

The induction of a wide spectrum of congenital malformations by maternal Zn deficiency was first demonstrated several decades ago, with early work mainly focused on dietary zinc deficiencies (Hurley and Swenerton, 1966). Rats can become zinc deficient within a few days of being fed a low zinc diet (Hurley and Swenerton, 1971). Clinical hallmarks of zinc deficiency include hyperkeratosis and/or parakeratosis of the stratified squamous epithelium of the stomach, esophagus, and feet, piloerection, reduced appetite, and reduced growth rate (Diamond et al., 1971). One mechanism of chemically induced maternal zinc deficiency involves inhibition of gastrointestinal absorption of zinc and/or enhanced urinary elimination. Most notable among compounds acting via this mechanism are the chelating agents that are used in industrial processes or as antidotes for metal intoxication. Administration of 2 or 3% ethylenediaminetetraacetic acid (EDTA) in the diet of pregnant rats produced signs of both maternal toxicity and teratogenicity, but these were not present in rats fed EDTA and 1000 ppm zinc. Interestingly, dietary administration of EDTA is more effective at inducing maternal and developmental toxicity than is EDTA given by gavage, consistent with the notion that EDTA inhibits gastrointestinal uptake of zinc by chelating the zinc present in the diet (Kimmel, 1977). Zinc deficiency is especially relevant to chemical safety evaluation because of a general response to toxic injury involving a cascade of events that can result in redistribution of zinc in the mother and, ultimately, embryonic zinc deficiency. Examples of developmental toxicants operating at least in part by this mechanism include 6-mercaptopurine, valproic acid, urethane, cadmium, ethanol, glucocorticoids, a-hederin, and lipopolysaccharide (Amemiya et al., 1989, 1986; Carey et al., 2000a; Keen et al., 1989, 2010; Leazer et al., 2002; Taubeneck et al., 1994). This common response to such a diverse set of agents is due to the role of zinc as a common effector molecule associated with the acute phase response to tissue damage or lipid peroxidation. Activation of the acute phase response triggers release of a number of cytokines, particularly tumor necrosis factor-a, leading to induction of hepatic metallothionein (MT). MT has the capacity to bind 7 mol of zinc or other divalent metal ions per mol of MT protein (Cousins et al., 2006). The degree of MT induction varies widely, but can reach as much as 100-fold under certain conditions (Daston, 1994). Maternal hepatic sequestration of zinc in turn leads to decreased distribution of zinc to the embryo. Importantly, teratogenic effects have resulted from a decrease in the transfer of zinc to the embryo, even in the absence of detectable decreases in absolute zinc levels (Daston et al., 1991a; Taubeneck et al., 1994) (Fig. 2). The effects of maternal zinc redistribution have been demonstrated following single acutely toxic doses or sustained dosing as carried out in safety studies. A study by Duffy and co-workers (Duffy et al., 1997) exemplifies the relative changes in zinc distribution and the resultant developmental effects that can occur. These investigators observed a 10-fold increase in maternal liver MT following a-hederin treatment of rats on GD 6–15, resulting in a 54% increase in maternal liver zinc, a 33% decrease in maternal blood plasma zinc, and a 70% decrease in the transfer of radiolabeled zinc to the embryo. Fetuses from a-hederin-treated dams had significantly lower body weights at term, and a threefold higher incidence of malformations. In studies with other MT inducers, a causal link between decreased zinc distribution to the conceptus and developmental toxicity was further substantiated by the amelioration of fetal effects via zinc supplementation of the diet, or in vitro by adding zinc to the serum used for whole-embryo culture (Daston et al., 1994). An essential role of MT in this zinc redistribution mode of action was confirmed by the lack of both hepatic zinc sequestration

Fig. 2

Zinc redistribution in response to maternal toxicity induced by diverse toxicants.

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93

and embryonic zinc deficiency in pregnant MT
/
knockout mice treated with a high dose of ethanol (Carey et al., 2000b). This mode of action has not only been demonstrated in rats and mice, but in rabbits as well (Pitt et al., 1997).

5.06.3.2.2

Folic acid deficiency

Folic acid (pteroylglutamic acid), along with choline, methionine, and homocysteine, is an essential player in one-carbon metabolism, and is well known as a dietary supplement used to protect against neural tube defects. Several drugs are known to alter maternal levels of folate by various maternally mediated mechanisms. For example, trimethoprim inhibits folate biosynthesis, sulfasalazine inhibits intestinal absorption of folate, and phenytoin may either inhibit folate absorption or interfere with folate transport (Hansen et al., 1997). These mechanisms can be distinguished from the actions of folate antagonists (e.g., aminopterin, methotrexate), which act directly on receptors in the embryo. Folate requirements during pregnancy are 5- to 10-fold higher than those in nonpregnant women and the high demand for folate must be met by dietary intake. In mice, dams fed a reduced folate diet (25% of optimal levels) for 2 months prior to mating had reduced fertility, increased resorptions, and smaller fetuses. However, the only developmental effect in dams given just slightly more folate (33% of optimal) was a reduction in fetal weight (Antony, 2007). Unlike the case of zinc, fetal folate levels tend to be given priority and are protected at the expense of maternal concentrations (Burren et al., 2008; McCann et al., 2006). This is due to an effective placental binding and transport system for folate.

5.06.3.2.3

Choline deficiency

Choline is critical in development as a precursor in the synthesis of acetylcholine, phosphatidylcholine, sphingomyelin, and platelet activating factor, and is required for metabolism of triglycerides (McCann et al., 2006). Maternal choline deficiency can be induced by a number of chemicals and is manifest by histological evidence of fatty liver, due to a lack of phosphatidylcholine, which then restricts the export of excess triacylglycerols from the liver, as well as muscle cell damage signaled by elevated creatine phosphokinase activity in serum. The demand for choline is especially high during pregnancy, but an efficient choline transport system is present that pumps choline against a concentration gradient, enabling depletion of maternal choline stores in an effort to maintain adequate choline in the developing conceptus. As a result, choline concentrations in fetal plasma are normally six- to sevenfold greater than those of maternal blood (Zeisel, 2006). Intrauterine choline deficiency is best known for its effects on the developing brain, resulting in effects on learning and memory. These behavioral changes are accompanied by electrophysiological, neuroanatomical, and neurochemical alterations that persist into adulthood (Kovacheva et al., 2007). Conversely, prenatal choline supplementation reportedly leads to enhanced memory and attention and protects against premature memory decline (Zeisel and Niculescu, 2006).

5.06.3.3

Maternal Cardiovascular Disturbances

Maternal cardiovascular conditions that have been studied for effects on development include maternal heart rate, uterine vasoconstriction, and various anemias. A number of these conditions appear to result in hypoxia to the embryo or fetus, although they can also impact fetal exchange of other gases, metabolic by-products, and nutrients. As with any developmental effect, the timing of the insult is a major determinant of toxicity. In the case of hypoxia-related changes, time dependence may reflect changing oxygen requirements between early pregnancy, when the embryo is adapted for a hypoxic environment, to fetal stages when oxygen demands are very high. An association between maternal bradycardia and cleft lip and palate was noted in A/J mice dosed with the anticonvulsant, phenytoin, although these effects were not seen in C57/BL6 mice (Watkinson and Millicovsky, 1983). Exposure of A/J mice to a hyperoxic atmosphere following treatment with phenytoin decreased the incidence of cleft lip and palate, leading the authors to surmise that bradycardia likely led to embryonic hypoxia as the proximate cause of teratogenesis (Millicovsky and Johnston, 1981). In a classic study of experimentally induced teratogenesis, Brent and Franklin (1960) clamped the uterine vessels of rats for up to 3 h on GD 9 and observed severe malformations as well as fetal death and growth retardation, which was found in subsequent work to be mainly attributable to hypoxia. Cocaine causes uterine vasoconstriction (Chasnoff et al., 1985), which is thought to be the mechanism behind a number of developmental effects (Plessinger and Woods, 1993). In rats, cocaine administration on GD 15 decreased uterine blood flow by > 50% (Lipton et al., 2002). Similar decreases in uterine blood flow in cocaine-treated sheep resulted in severe fetal hypoxia. However, there was no change in fetal oxygenation when cocaine was injected directly into the fetus, indicating a maternally mediated mode of action (Woods et al., 1987). Similarly, injection of adrenaline or vasopressin into pregnant rats produced uterine vasoconstriction and signs of fetal hypoxia (Chernoff and Grabowski, 1971; Clark et al., 1984). Certain chemical exposures can result in various anemias, either through decreased red cell production or increased loss of red cells (e.g., hemorrhage, hemolysis). The relationship between maternal hemolytic anemia and teratogenesis was investigated in a study with diflunisal, an anti-inflammatory analgesic. In a standard developmental toxicity study in rabbits, diflunisal caused axial skeletal malformations, but only at doses that also caused a severe (> 50% decreased hemoglobin and erythrocyte count) maternal hemolytic anemia (Clark et al., 1984). To determine if the maternal anemia was responsible for the fetal malformations, a subsequent experiment was performed in which a single dose of diflunisal was given on GD 5. This treatment caused an anemia persisting until GD 15 and axial skeletal malformations, yet the drug was cleared from the maternal circulation prior to GD 9. As GD 9 is the critical day for induction of axial skeletal malformations by hypoxia treatment, the authors concluded that maternal anemia was the probable teratogen.

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With anemias, it appears that they must be severe in order to adversely impact in utero development. Slight to moderate anemias seen in pregnant rats and/or rabbits exposed to propylene dichloride (Kirk et al., 1995) or ethylene glycol monobutyl ether (Tyl et al., 1984) were not teratogenic, although they were associated with minor delays in skeletal ossification. In sheep, maternal hematocrit had to decline > 50% before fetal oxygen consumption was affected (Paulone et al., 1987).

5.06.3.4

Hyper- and Hypoosmolality

The principle of osmosis is central to all of biology, yet the potential osmotic load associated with administration of high doses of small molecules is rarely considered in toxicology. In addition to the direct hyperosmotic effects of chemical exposure (Glasser et al., 1973), hyperosmolality can also result indirectly from dehydration, increased salt intake, loss of body water through vomiting, or inhibition of water reabsorption in the renal collecting duct (e.g., diabetes insipidus). Conversely, compounds such as morphine, nicotine, certain tranquilizers, and anesthetics increase water reabsorption in the kidney and thus have the potential to induce hypoosmolality. As with acid–base balance, there are many maternal systems that tightly regulate extracellular fluid osmolality, among them the thirst centers in the brain that affect water intake, and arginine vasopressin that controls reabsorption of water in the renal collecting ducts. At the cellular level, sodium and chloride ion concentrations are the major determinants of osmotic pressure. Proteins in plasma and interstitial fluid are also an important source of osmotic pressure, usually referred to as oncotic or colloid osmotic pressure. This is particularly important in the movement of solutes across capillary walls. Studies on preimplantation embryos have demonstrated increased expression of genes such as p38 MAPK and CCM2 in response to hyperosmotic environments (Fong et al., 2007), although we know that embryos at these early stages can be very sensitive to changes in osmolality. Limited data are available concerning osmoregulation during organogenesis, although one study (Carney et al., 1996) showed that GD 10.5 rat conceptuses cultured with intact visceral yolk sac placenta grew normally in culture medium that was extremely hypertonic (418 mOsmol kg
1 H2O, vs 299 for control medium). The visceral yolk sac placenta may play a crucial role in the maintenance of embryonic osmolality. For example, trypan blue treatment of embryos in vivo and in vitro caused lysosomal damage to the visceral yolk sac endoderm, a decrease in exocoelomic fluid osmolality, and a corresponding increase in the incidence of embryonic edema (Rogers et al., 1985). The opposite effect was observed following treatment with leupeptin, an inhibitor of yolk sac lysosomal proteinase activity, which caused an increase in exocoelomic fluid osmolality and malformations characterized by decreased neural tube volume (Daston et al., 1991b). Embryonic edema, blistering, and hemorrhage are common responses to many teratogenic agents, suggesting that the embryo may have a limited ability to cope with changes in osmolality. Grabowski (1966) coined the term “edema syndrome” for this mode of teratogenicity, which he first observed in chick embryos subjected to hypoxia. During early fetal stages, injection of hypotonic or hypertonic solutions into the mother brings about parallel changes in maternal and fetal plasma osmolality (Bruns et al., 1964; Dancis et al., 1957). The consequences of these changes are unknown, although studies in pregnant sheep suggest that prolonged fetal hyperosmolality (induced by maternal water deprivation) may imprint the hypothalamic–pituitary arginine vasopressin regulatory system (Ramirez et al., 2002). Once the fetal kidneys become functional, osmotic challenges can be dealt with by concentration or dilution of the fetal urine and excretion of this urine into the amniotic cavity.

5.06.3.5

Altered Body Temperature

Elevations in maternal core body temperature of approximately 3–5 C have been demonstrated in a number of animal species to induce neural tube defects, axial skeletal malformations, and other developmental problems. Severe hyperthermia may also be developmentally toxic in humans, although this issue has been difficult to resolve due to the coexistence of underlying disease conditions responsible for the hyperthermia. As the issue of hyperthermia during pregnancy has been extensively addressed by others, readers are referred to several reviews on this subject (German, 1984; Mirkes, 1985). Decreased body temperature can be an unrecognized response to systemic toxicity, particularly in very small laboratory animals such as mice (Spencer et al., 2007). The hypothermic response, which is brought about by decreasing heart rate and blood pressure and increasing peripheral blood flow (i.e., increased heat dissipation), apparently confers a survival advantage relative to animals in which the response was blocked by various experimental means (Watkinson and Gordon, 1993). The hypothermic response has been studied only to a limited extent in pregnant animals, and results are contradictory. Early work on the effects of severe hypothermia during pregnancy showed increased resorptions and morphological abnormalities (Munro and Barnett, 1969), while mouse whole-embryo cultures maintained at 32 or 35 C exhibited decreased growth and protein content, incomplete neural tube closure, poor brain expansion, and rotational abnormalities (Smoak and Sadler, 1991). Conversely, Randall et al. (1988) failed to find a difference in fetal malformations or fetal body weight in GD 10 mice exposed to a developmentally toxic and hypothermia-inducing dose of ethanol, followed by housing at 32 C (prevented hypothermic response) versus room temperature (control). Yet another set of studies demonstrated a protective effect of hypothermia. For example, the incidence of malformations caused by radiation exposure (Marois, 1966) or uterine vascular clamping (George et al., 1967) was significantly reduced by maternal hypothermia. In mouse whole-embryo cultures, hypoglycemic serum was less teratogenic when the cultures were maintained at 32 or 35 C versus 37 C (Smoak and Sadler, 1991).

Maternally Mediated Developmental Toxicity

5.06.4

95

Data Evaluation: Unraveling Maternal and Developmental Toxicity Findings

Assuming that testing protocols continue to require the inclusion of high, overtly toxic doses to pregnant laboratory animals, we can expect that approximately 75% of chemicals evaluated in these tests will show developmental effects only at maternally toxic doses (Khera, 1985). While such descriptive data can be used in conventional risk assessments, relying only on these data is problematic due to the number of assumptions that must be invoked when extrapolating the animal data to assess human risk. A preferable, albeit more demanding, approach would be to understand the basic chain of events or mode of action, starting from entry of the chemical into the maternal system, to disposition into maternal, placental, and embryonic compartments, followed by the essential pharmacodynamic changes that lead to toxicity. In the context of the maternal toxicity–developmental toxicity relationship, the critical goal is to determine whether or not the perturbation of maternal physiology is an essential requirement for developmental toxicity, such that the absence of the maternal perturbation would ensure, with a high degree of confidence, that the embryo/fetus is safe from harm (Fig. 3). How one might design experiments to determine whether developmental toxicity is due to direct effects of the chemical on the embryo or is mediated by an effect on the mother is exemplified by the studies on cocaine and diflunisal discussed above, and by studies on 2-amino-2-methylpropanol (AMP) (Ball et al., 2014). In female rats, oral dosing with AMP starting several weeks prior to mating resulted in maternal hepatotoxicity and embryo resorption. Additional studies demonstrated that dosing had to begin at least two weeks prior to mating and through to the period of implantation, that there was a steep dose response, and that the toxicity was “all or none,” in that resorption occurred very early (gestation days 6–8) or not at all, with surviving embryos developing normally. These findings suggested a window of susceptibility that began in the dam prior to mating, and that a threshold event might be responsible for the embryo loss. Along with the “all-or-none” early resorptions, this led the investigators to suspect a maternally-mediated mode of action. Exposure of organogenesis stage embryos to AMP or to serum from AMP-treated rats in whole embryo culture produced no embryotoxicity, further indicating that the in vivo post implantation loss following AMP exposure was due to an effect on the mother that interfered with her ability to support implantation. The final study in this series examined effects of AMP on uterine histology. AMP treated dams exhibited vacuoles in the uterine luminal epithelium in decidual swellings on gestation day 6, with more severe vacuolation in sites adjacent to embryos appearing to be undergoing resorption. Gene expression analysis of the decidual swellings demonstrated reduced expression of genes associated with formation of tight junctions,

Fig. 3

Decision logic for assessing the human relevance of a maternally mediated mode of action (MOA).

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structures important for development of the decidual epithelial barrier function thought to protect the early embryo. This series of experiments supported the hypothesis that AMP exerts its effect on the mother, affecting the ability of the uterus to support implantation, possibly due to an effect on the decidual epithelium. The potential role of maternal hepatotoxicity was not ruled out by these studies and remains to be determined. The examples cited in this chapter constitute some of the more common profiles of maternal toxicity that might be encountered as a result of chemical toxicity, and are instructive for interpreting data on the maternal toxicity–developmental toxicity relationship. Discussed below are some basic considerations that can be used in a weight of evidence evaluation of data. 1. Biological plausibility of a causal link. The type of developmental effect should be commensurate with the nature and severity of maternal toxicity. Examples General decrease in maternal food consumption and body weight gain; decreased fetal body weight. Plausible (see section on Maternal Weight Gain and Food Intake, and Malnutrition). General decrease in maternal food consumption and body weight gain; fetal malformations. Unlikely (see section on Maternal Weight Gain and Food Intake, and Malnutrition). Extensive maternal tissue damage, MT induction, and resultant zinc redistribution; fetal malformations. Plausible (see section on Zinc deficiency). Maternal stomach and esophagus show microscopic evidence of hyper- and parakeratosis following dosing with a chelating agent; fetal malformations. Plausible (see section on Zinc deficiency). Mild maternal metabolic acidosis (plasma pH 7.2, 20% decrease in PCO2 and bicarbonate); neural tube and eye malformations. Unlikely (see section on Acid-Base Imbalance). Maternal red blood cell (RBC) count decreased by 15%, slight increase in spleen weight; fivefold increase in embryo/fetal deaths. Unlikely (see section on Maternal Cardiovascular Disturbances). Maternal RBC count decreased by 15%, slight increase in spleen weight; slight decrease in fetal body weight. Plausible (see section on Maternal Cardiovascular Disturbances). 2. Temporal correspondence. In development, timing is everything! Therefore, the timing of maternal toxicity should correspond with the estimated stage of gestation when development was affected, based on knowledge of embryo development. Examples Significantly decreased body weight during the last week of gestation (rat); decreased fetal body weight and reduced ossification of the calvarium. Plausible (see section on Maternal Weight Gain and Food Intake, and Malnutrition). (See also Carney and Kimmel (2007) for profiles of delayed skeletal ossification.) Significantly decreased body weight during the first three days of dosing (GD 7–10 in a rabbit) with recovery to normal body weight by GD 14; decreased fetal body weight. Unlikely (see section on Maternal Weight Gain and Food Intake, and Malnutrition). Maternal body weight loss during the last five days of gestation; fetal anophthalmia. Unlikely due to the fact that the eyes form very early during organogenesis, whereas the maternal insult occurred late in the fetal period. 3. Individual animal correspondence. Assessment of individual animal data is critical in these evaluations, particularly with rabbits owing to their typically greater degree of interanimal variability. The greater the correspondence between severity of maternal toxicity in individual maternal animals and effects in their litters, the greater the weight of evidence supporting the association. An example of this type of analysis can be found in a study of the herbicide triclopyr butoxyethyl ester in rats (Carney et al., 2007) in which the majority of developmental effects were restricted to just three dams that exhibited an especially high degree of maternal toxicity at a time in gestation that was consistent with the type of defects observed. The remaining 18 dams showed very little maternal toxicity and produced no evidence of developmental toxicity. 4. Potential interactions. Another consideration is that multiple stressors can impact upon maternal physiology to heighten the overall maternal response and exacerbate secondary developmental effects. Studies in rodents have shown that stress hormones may act as potentiators, rather than direct developmental toxicants (Goldman and Yakovac, 1964; Hartel and Hartel, 1960; Rasco and Hood, 1994). Interactions should also be considered when applying food restriction data to the interpretation of toxicity studies, as food restriction adequately models the impact of reduced maternal body weight, but not the stress or malaise associated with target organ toxicity. Thus, on an actual toxicity study, the combined impact of reduced maternal body weight gain and maternal stress on the developing fetus could be greater than suggested by food restriction data alone. 5. Mode of action data. For studies that match some of the common profiles of maternal and developmental effects described in this chapter, information from the literature may be sufficient to conclude that there is a plausible causal relationship between maternal toxicity and developmental toxicity. However, other cases will warrant the generation of mode of action information directed at determining human relevance (Seed et al., 2005). Whole-embryo culture can be a powerful tool in delineating between maternally mediated versus direct modes of action, as the test compound or its metabolites can be added directly to the cultures, thus removing the impact of maternal influences. Conversely, to evaluate the impact of maternal factors such as nutrient deficiencies or altered serum biochemistry, an ex vivo embryo culture approach can be used in which untreated, naive embryos are grown in serum from a separate group of treated animals (Chatot et al., 1984). These in vitro strategies can be complemented with more detailed evaluations of maternal physiology, which now include possibilities like wireless telemetry monitoring.

Maternally Mediated Developmental Toxicity

5.06.5

97

New Concepts in Maternal Toxicity for Safety Evaluation

Despite many workshops and symposia focused on the relationship between maternal toxicity and developmental toxicity, the same basic issues have remained largely unresolved. The present assemblage of knowledge concerning the specific details of different types of maternal physiological perturbations and their impact on development should be useful in resolving at least some of these issues. There is, however, another way to deal with the vexing issue of maternal toxicity and developmental toxicity, and that is to avoid excessive maternal toxicity altogether. Alternative approaches to determining dosage ranges for developmental toxicity studies have been discussed, including those based on maternal pharmacokinetics (Carney et al., 2011) and use of a highest dosage not exceeding the maternal NOAEL (Giavini and Menegola, 2012). As mentioned previously, developmental toxicity studies have historically included administration of maximally tolerated doses, most commonly given as an oral bolus (gavage). Quite often gavage dosing results in very high peak blood levels that are not relevant to human exposures, which occur by different routes of exposure and which may be spread over a longer period of time, rather than incurred all at once (Carney et al., 2001). In many cases, particularly for agricultural and industrial chemicals, the dose levels tested in animals are several orders of magnitude higher than those to which people might be exposed. There is a trend in the field of toxicology to gradually move away from extremely high dose testing toward doses and routes of exposure that more accurately mimic actual human exposure levels. This trend toward lower, more realistic dose levels is being driven by several factors, namely, (1) the availability of more sensitive endpoints (e.g., gene expression), (2) animal welfare concerns about excessive pain and distress, and (3) recognition that high doses often bring about nonlinear shifts in pharmacokinetics, which often are not relevant to human exposures. The third point is particularly intriguing, as dose-dependent nonlinearities such as those related to saturation of metabolizing enzymes can result in a different spectrum of metabolites being tested at high versus low doses. Thus, a paradigm shift in dose level selection practices may be the best solution to the long-standing problem of maternal toxicity in safety studies, as it would avoid the issue altogether.

See also: 5.07. Paternally Mediated Developmental Toxicity. 5.08. Epigenetics and the Developmental Origins of Health and Disease.

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Further Reading Altobelli, G., Bogdarina, I. G., Stupka, E., Clark, A. J., & Langley-Evans, S. (2013). PloS One, 8, e82989. Daniel, Z., Swali, A., Emes, R., & Langley-Evans, S. C. (2016). Genes & Nutrition, 11, 27. Engeham, S. F., Haase, A., & Langley-Evans, S. C. (2010). The British Journal of Nutrition, 103, 996–1007. Engeham, S., Mdaki, K., Jewell, K., Austin, R., Lehner, A. N., & Langley-Evans, S. C. (2012). Journal of Nutrition and Metabolism, 2012, 989037. Swali, A., McMullen, S., Hayes, H., Gambling, L., McArdle, H. J., & Langley-Evans, S. C. (2011). PloS One, 6, e23189. Swali, A., McMullen, S., Hayes, H., Gambling, L., McArdle, H. J., & Langley-Evans, S. C. (2012). PloS One, 7, e48133.