13 Vincent F. Garry and Peter L. Truran There is increasing recognition that early life stages, including fetal development, can be particularly vulnerable to adverse effects from drugs and chemicals. During early life, toxicity from even transient exposures can be profound, long-lasting, and in some cases, trans-generational. Developmental toxicity focuses on the study of birth and developmental anomalies associated with pharmacologic and environmental agents, and the underlying biochemical and molecular mechanisms. This chapter discusses: Josef Warkany (1902–1992) is described by his peers as the father of modern teratology as a clinical and experimental discipline. His life’s work grew from discoveries of the causal role of nutritional and vitamin deficiencies in birth defects, to explorations of the teratologic effects of drugs (warfarin, salicylates, thalidomide, methotrexate, and aminopterin). His ideas were merged with those of James G. Wilson, who formulated the central working paradigm of teratology in the twentieth century, and which is defined as the principles of teratology. In the period from 1900 through the 1960s, experimental embryology came to the forefront. Developing embryos in sea urchins, amphibians, and chicks were painstakingly dissected, and embryonic tissues at early stages of development were subjected to experimental manipulation. Studies by Weismann, Spemann, and Horstadius provided precious insights into cellular development and organ system differentiation. In parallel with these studies, Horstadius and others explored the effects of chemicals and alterations in the physical environment of the developing embryo. The six principles of teratology put forward by James G. Wilson (1915–1987) integrated the knowledge gained from developmental biology, genetics, clinical medicine, and biochemistry to describe the major factors that may be involved in a teratologic event. These principles, which continue to be relevant today, are as follows: More than 3 in every 100 live births will result in a child with a major structural or genetically based birth defect. Birth defects are a major contributor to infant mortality and result in billions of dollars in health care costs. In the United States, national estimates of birth defect prevalence are based on birth certificate data. Based on these data, the overall birth defect rate per year from 1999 to 2005 was approximately 1 in 100 live births. However, birth certificate data underestimate the frequency of birth defects, since not all birth defects are apparent and reported at birth or shortly thereafter. To illustrate the point, the relative rate of birth defects in a cohort of members of the farming community reported in the first year of life, were examined. Comparison of birth defects rates confirmed in medical records versus those from birth certificate data demonstrated that the relative rate per 1000 births of CNS (4.6 vs. 1.3), musculoskeletal (9.8 vs. 6.5), and cardiovascular (5.2 vs. 3.7) birth defects were higher in the medical record confirmed data set. On the other hand, gastrointestinal (2.0 vs. 1.3), urogenital (4.0 vs. 4.4), and genetic/metabolic (0.65 vs. 1.7) birth anomaly rates were nearly equivalent. It is apparent from these data that, although birth anomalies visible at birth or symptomatic shortly thereafter can be captured in birth certificate data, many birth and developmental anomalies make their appearance well after the newborn period. For example, in the same study, 62% of the birth anomalies reported were identified in the first year of life, another 10% within years 1–3, and 20% in children more than 3 years old. More comprehensive work is ongoing through the Centers for Disease Control (CDC). For more than 30 years metropolitan Atlanta, Georgia, through the CDC, has engaged, and continues to develop, an active birth defect reporting system. Medical records of each live birth in the five counties of metropolitan Atlanta are examined and updated through age 5 by trained abstractors. Major structural and or genetic defects are identified in this group. From 1978 to 2005 the overall prevalence of major structural defects was stable, varying from 2.8 to 3.0 per 100 live births. Male children had more defects than females; fewer birth defects were observed among minorities. Issues such as access to care and poverty appear unresolved in these assessments. Active reporting of birth defect prevalence nationwide is limited to specific major anatomic disorders. However, the cause(s) of approximately 70% of birth defects reported are unknown, and there are limited data available on the frequencies of genetic, environmental, infectious, and medication-related causes of birth defects. The spectrum of agents known to cause human birth defects is broad. A comprehensive catalogue of known human teratogenic agents was assembled by Shepard in order to capture the available knowledge base regarding agents linked to human birth defects. Nevertheless, based on our current assessments, it is likely that both the spectrum of agents and frequency of birth defects are wholly underestimated, particularly in relation to workplace exposures. Given the numbers of women employed outside of the home, it is appropriate to make an assessment of pregnancy and reproductive health issues related to the workplace. We will also explore adverse birth and developmental risk. Pregnancy is a complex physiologic condition that involves dramatic changes in the developing fetus and placenta, and in the cardiovascular, neuroendocrine, renal, respiratory, and metabolic systems of the pregnant woman. All these changes take place in concert as each trimester of pregnancy proceeds. In this milieu, xenobiotic chemical and pharmacologic agents, and physical and psychological stressors, have unique opportunities to exert adverse effects through dysregulatory and cytotoxic mechanisms. The first trimester is the time of structural development of major organ systems of the embryo. The timing of cell movements, and the homing of cells to their developing organ sites, involves exquisitely integrated morphogenic events. For example, formation of the kidney involves bringing together the structural elements of the glomeruli, the parenchyma of the organ, along with the formation of renal tubules and the collection system of the kidney. As pregnancy proceeds, the fetal kidney develops and evolves along phylogenetic lines from the primitive pronephric kidney, through the mesonephric kidney, to the mature metanephric kidney. Accordingly, this period represents a time of unique sensitivity to relatively low-dose chemicals, structural teratogens, whose mechanism of action will interfere with development and differentiation of the organ systems. During the first 4–6 weeks of gestation there are dramatic changes in maternal hemodynamics including increased cardiac output, expansion of the plasma volume, and reductions of vascular resistance and arterial pressure. By late pregnancy, the volume of circulating blood has increased by 40–45%. Vascular resistance remains low due to reduced sensitivity to angiotensin and increased nitric oxide levels. The end result is a relative lowering of serum albumin and other serum protein levels. As a consequence, the ratio of protein-bound drugs/toxicants to free drug/chemical levels is altered; more free-drug/toxicant is then available for hepatic biotransformation and/or renal excretion. The metabolic activities of many of the P450 enzymes are increased during pregnancy. In particular, among the 57 gene products responsible for the expression of the broad range of P450 enzymes, cytochrome P450 3A4 (CYP3A4) is induced to higher activity. This enzyme plays a major role in the metabolism/detoxification of a wide variety of xenobiotics, including approximately 50% of therapeutic drugs. Increased transformation of xenobiotics, toxins, and chemicals with mutagenic potential results in their rapid clearance from the maternal circulation, and hence lessens the amount of toxic agent available for fetal exposure. The metabolic activities of other P450 enzymes (CYP2D6, CYP2A6, CPY2C9) are similarly enhanced. For example, metabolism and clearance of both nicotine and cotinine are enhanced during pregnancy through increased CYP1A2 activity. The metabolic activity of some other P450 enzymes including CYP1A2 (e.g., caffeine metabolism) and CYP2C19 are reduced during pregnancy. The liver of the developing fetus also contributes to CYP450-mediated metabolism. Among the CYP450 enzymes present in fetal liver, CYP3A7 appears early in development and is thought to play a major role in fetal metabolism of xenobiotics including the metabolic activation of Aflatoxin B1. It is noteworthy that, immediately after birth, CYP3A becomes the predominate isoform in the metabolism of xenobiotics. During pregnancy, the human placenta is an important endocrine organ. Placental metabolic capacity is mainly directed toward synthesis and metabolism of hormones (both steroidal and protein). Smoking, xenobiotics such as TCDD, and other endocrine disruptors are thought to affect placental steroidogenesis. As the third member of the CYP450 xenobiotic metabolic triumvirate, the trophoblastic placenta is believed to play a fetoprotective role in the first trimester of pregnancy. CYP1A1 is the only placental xenobiotic metabolizing enzyme whose expression and inducibility have been demonstrated throughout pregnancy. Products of cigarette smoking and the pro-carcinogenic PAHs undergo bioactivation by CPYA1. Elevated CYPA1 activity has been associated with adverse birth outcomes such as premature birth, intrauterine growth retardation, and structural abnormalities. Minute ventilation (FEV1) is increased by the 7–8 weeks pregnancy with an increased tidal volume of 40% and decrease residual volume of 20%. This increased respiratory rate, accompanied by higher cardiovascular output, leads to enhanced pulmonary absorption of air pollutants, including cigarette smoke. More importantly, adaptation of the respiratory system provides enhanced oxygenation for prospective mother and fetus. The renal glomerular filtration rate begins to increase in the first trimester of pregnancy and peaks in the second half of pregnancy at a level 40–60% higher than nonpregnant women. An increased excretion of low-molecular-weight water-soluble toxicants can be expected. The extraordinary changes of pregnancy are designed to assure development and survival of the fetus through physiologic adaptation of the major organ systems and metabolism of the prospective mother. At the same time, these postconception physiologic changes can constitute individual and collective risk factors for teratologic and other adverse reproductive effects caused by chemical and other stressors. According to the U.S. Department of Labor statistics (2010), about 59% of women residing in the United States work for pay outside the home. Nearly three of every four women who are employed are of reproductive age. According to one source, fully 80% of working women will become pregnant during their working lives. Approximately, 40% of women who were, or became, pregnant while working remained in the workforce. In fact, a woman’s right to be pregnant in the workplace is protected by law. As an extension of the Title VII Civil Rights Act (1964), the Pregnancy Discrimination Act of 1978 stipulated that all employers treat pregnant and nonpregnant employees in the same way, both in terms of benefits received and in all other respects. The Family and Medical Leave Act (FMLA) 1993 further stipulated that men and women might take as many as 12 weeks of unpaid leave annually for the birth or adoption of a child. In other countries (European Union, Canada), family leave includes financial and social support. Given the breadth and scope of this demographic information, it is remarkable that there are so few concrete human data on the risks for birth defects in occupations where women of reproductive age are employed and exposed to toxicants. According to epidemiologic reviews, convincing evidence linking occupational exposure during pregnancy and birth defects is lacking. It is not surprising, therefore, that in the occupational health sector and in the legal arena, strategies and actions devoted to birth defect prevention are limited. In guidance provided by the American College of Occupational and Environmental Medicine indicates, needs assessment points to the application of technologies, including medical surveillance, biomarker exposure/risk assessments, and population-based epidemiology in order to detect persons at risk for birth defects. These are key elements in birth defect preventative strategies but, to date, progress toward their implementation has been notably short of ideal. Nevertheless, certain maternal occupations can be established as risk factors for birth defects. Data from the national birth defects prevention study show that women employed in any one several occupational groups (janitors/cleaners, scientists, and electronic equipment operators) have an excess risk for birth defects. Other published works provide some detail regarding exposure to chemical agents linked to birth defects. Children born to nurses exposed to anesthetic gases may be at increased risk for birth defects, as are those handling cytostatic agents. In general, nurses may have a modest excess risk. Solvent exposure in the workplace, mainly for those employed as painters, has been associated with increased risk of birth defects. For males, employment as a painter 3 months prior to spouse’s pregnancy was associated with increased risk of birth defects. For females, exposure to solvents in the first trimester of pregnancy has been associated with increased risk of bearing children with cardiac birth defects. Solvents and other products classified as endocrine-disrupting chemicals can be risk factors for male genital malformations. Surprisingly, genetic studies of the offspring of atomic bomb survivors (Hiroshima/Nagasaki, Japan) conducted since 1948 have yielded little or no evidence of excess birth defects. Future molecular genetic studies of the survivors are expected to yield information on transgenerational changes in the genomes of their offspring including changes affecting adult-onset multifactorial diseases, e.g. diabetes. In studies of chronic low-level radiation, such as occurs in the nuclear industry, women radiation oncologists exposed during their pregnancy show little or no difference in the frequency of birth defects. One small study, where maternal occupational exposure to radiation occurred in the first trimester of pregnancy, suggests a significant excess of birth defects in children from radiation-exposed mothers compared to unexposed mothers. In contrast to available data on humans, animal studies precisely document radiation doses, timing during pregnancy, and types of birth defects that may occur. Finally, rules for radiation dose limits apply once a pregnant employee declares her pregnancy. The code of federal regulations set an occupational exposure limit of 0.005 Gy (0.5 rad). Despite the limited demonstration of birth defects causally linked with occupational exposures, the possibility of such effects remains a concern. Clarification of current occupational epidemiologic data, with continued data collection on pregnancy, occupation, and the utilization of the newer pharmaco-genetic approach, will yield concrete evidence-based advice to the community at large. Most human teratogens have been identified by astute clinicians and/or through epidemiologic study. That being said, animal studies historically have been, and are currently, the mainstay of nonclinical investigative drug development and environmental chemical studies of reproductive toxicity. The typical approach to animal chemical teratogenicity assessment is shown in Figure 13.1. In this scheme test chemical is administered to the dam at the time of implantation through the period of organogenesis and organ system development. Necropsy is conducted on the days specified, adjusting for gestation time per species. This is but one part of the developmental toxicologic testing proscribed by U.S. Federal Regulation (Title 40 subpart H 799.9370 TSCA prenatal developmental toxicity) and by OECD guidelines (Guideline 414 prenatal toxicity study). On the whole, both regulatory program assessments are quite similar. They are highly detailed in their study requirements. We will limit our discussion to the basic requirements of the test: Figure 13.1 Scheme for chemical teratogenicity assessment. G, gestation; GD, gestational day; L, lactation; M, mating; N, Necropsy; Q, quarantine; W, wean. Despite the detailed study protocol briefly recounted here, the overall probability of predicting a human teratogen in standard animal (rat, rabbit) reproductive studies is only moderate (Table 13.1). Increasing the number of species tested (upward of five or more) improves the overall capture rate to almost 100% positive correlation with known human teratogens. While the possibility of animal-based precise predictive developmental toxicology exists, the overall economic and ethical costs are prohibitive. Table 13.1 Predictive Value of Animal Teratogenesis Studies of Known Human Teratogens (N = 35 Schradein 1993)
DEVELOPMENTAL TOXICOLOGY
13.1 BIRTH AND DEVELOPMENTAL ANOMALIES: TERATOLOGY
The Principles of Teratology
Birth Defect Prevalence
13.2 PHYSIOLOGY OF PREGNANCY
Maternal Cardiovascular System
Metabolism in the Prospective Mother
Metabolism in the Fetus
Metabolism in the Placenta
Maternal Respiration
Maternal Renal Function
Summary of Pregnancy Physiology
13.3 WORKPLACE, OCCUPATION, AND ADVERSE REPRODUCTIVE EFFECTS
Nursing/Health Care Workers
Maternal and Paternal Exposure to Organic Solvents
Radiation
Perspective from Studies of Pregnant Workers
13.4 GENERAL TESTING METHODS FOR TERATOGENIC EFFECTS IN EXPERIMENTAL ANIMALS
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