10

Effects on Veterans’ Descendants

Chapter Overview

Based on new evidence and a review of prior studies, the committee for Update 2014 did not find any new significant associations between the relevant exposures and adverse outcomes in future generations. Furthermore, the committee has changed the previous categorization of exposure to the chemicals of interest (COIs) and spina bifida from limited suggestive to inadequate or insufficient, consistent with all other birth defects and parental exposures to the COIs. Current evidence supports the findings of earlier studies that

• No adverse outcomes in future generations had sufficient evidence of an association with the COIs.
• There is inadequate or insufficient evidence to determine whether there is an association between parental exposure to the COIs and birth defects, childhood cancers, or disease in their children as they mature or in later generations.

The original report in this series, Veterans and Agent Orange: Health Effects of Herbicides Used in Vietnam¹ (VAO; IOM, 1994) contained a single chapter devoted to reproductive outcomes, as was the case through the publication of

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¹Despite loose usage of “Agent Orange” by many people, in numerous publications, and even in the title of this series, this committee uses “herbicides” to refer to the full range of herbicide exposures experienced in Vietnam, while “Agent Orange” is reserved for a specific one of the mixtures sprayed in Vietnam.

Veterans and Agent Orange: Update 2000, hereafter referred to as Update 2000 (IOM, 2001). (Analogous shortened names are used to refer to the updates for 1996, 1998, 2002, 2004, 2006, 2008, 2010, and 2012 [IOM, 1996, 1999, 2003, 2005, 2007, 2009, 2011, 2014].) In Update 2002, the chapter’s concerns were extended to include the consideration of developmental effects. In Update 2008, the chapter also addressed the possibility that adverse effects of exposure to the chemicals in the herbicides used by the military in Vietnam might extend beyond the children of exposed people and affect future generations.

The committee for Update 2012 decided to divide the material into two separate chapters. Chapter 9 contains information on reproductive outcomes affecting the parental generation and the course of gestation. This chapter focuses on the potential adverse health outcomes in the first generation and on issues related to the possibility of adverse effects occurring in even later generations. Adverse health outcomes due to parental exposure that demonstrate their ongoing heritability by being transmitted to grandchildren and beyond are termed transgenerational effects. Since its inception, the VAO series has considered birth defects (primarily limited to problems detectable at birth or within the first year of life) and childhood cancers (usually restricted to particular cancers that characteristically appear in infants and children and are diagnosed before the age of 18 years). Because of concerns increasingly expressed by veterans and a corresponding interest in the Department of Veterans Affairs, in Update 2010 the attention of the VAO committees was extended to include all types of medical issues occurring in the veterans’ children regardless of age and to include such problems in successive generations. The research community has made considerable progress in understanding the processes that may result in such delayed outcomes, often referred to as the Developmental Origins of Health and Disease (DOHaD). It is hoped that by devoting a separate chapter to the potential problems for the progeny of Vietnam veterans—and perhaps their descendants—we can more clearly present the evidence for maternally and paternally mediated effects separately, because the underlying biology is quite distinct in the two cases.

This chapter summarizes the scientific literature published since Update 2012 that investigated associations between parental exposure to herbicides and adverse effects on offspring, including future generations, throughout their lifespans. The epidemiologic literature considered in this chapter includes studies of a broad spectrum of effects in the children of Vietnam veterans or other populations occupationally or environmentally exposed to the herbicides sprayed in Vietnam or to the contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Because some polychlorinated biphenyls (PCBs), some polychlorinated dibenzofurans (PCDFs), and some polychlorinated dibenzodioxins (PCDDs) other than TCDD have dioxin-like biologic activity, studies of populations exposed to PCBs or PCDFs were reviewed if their results were presented in terms of TCDD toxic equivalents (TEQs). Although all studies reporting TEQs based on PCBs were reviewed, those studies that reported TEQs based only on mono-ortho

PCBs (which are PCBs 105, 114, 118, 123, 156, 157, 167, and 189) were given very limited consideration because mono-ortho PCBs typically contribute less than 10 percent to total TEQs, based on the World Health Organization (WHO) revised toxicity equivalency factor (TEF) of 2005 (La Rocca et al., 2008; van den Berg et al., 2006). Although some multigenerational studies have been conducted on laboratory animals, to date there have not been human studies of descendants beyond the first generation for the chemicals of interest (COIs).

Because most Vietnam veterans are men, the primary focus of the VAO series has been on the potential adverse effects of herbicide exposure on men. For nonreproductive outcomes, the etiologic importance of an exposed person’s sex does not play a dominant role; but for consideration of the biological mechanisms for the possible transmission of adverse effects to future generations, the gender of the exposed individual is critically important, as discussed below. About 8,000 women served in Vietnam (H. Kang, US Department of Veterans Affairs, personal communication, December 14, 2000), so adverse outcomes in the offspring of female Vietnam veterans are a concern. Exposure scenarios in human populations and experimental animals studied differ in their applicability to our population of concern according to whether the exposed parent was male or female, and it is necessary to evaluate the effects of maternal and paternal exposure separately. As will be noted repeatedly, however, almost all Vietnam veterans were men, but the amount of research providing reliable information on the consequences of paternal exposure is extremely sparse for the COIs in the VAO report series and also for the full array of environmental agents that may pose threats to the health of future generations.

In addition, for published epidemiologic or experimental results to be fully relevant to evaluation of the plausibility of reproductive effects in Vietnam veterans, whether female or male, the veterans’ exposure needs to have occurred before the conception of the children. With the exception of female veterans who became pregnant while serving in Vietnam, pregnancies that might have been affected occurred after deployment, when primary exposure had ceased. In the case of pregnancies of women who have previously been substantially exposed to the lipophilic dioxins, the direct exposure of the fetus throughout gestation is possible through the mobilization of toxicants from the mother’s adipose tissue. In contrast, adverse effects on offspring mediated by male veterans would be via alterations in the sperm genome and associated ribonucleic acids (RNAs) or semen that would have been transmitted after exposure and deployment.

The categories of association and the approach to categorizing the health outcomes are discussed in Chapters 1 and 2. To reduce repetition throughout the report, Chapter 6 characterizes the study population and presents design information on new publications that report findings on multiple health outcomes or that revisit study populations considered in earlier updates.

BIOLOGIC PLAUSIBILITY OF EFFECTS
IN VETERANS’ DESCENDANTS

Few offspring studies of the four herbicides in question have been conducted, particularly for picloram and cacodylic acid, and those studies generally have shown toxicity only at very high doses. Thus, the preponderance of the following discussion concerns TCDD exposures, which outside controlled experimental circumstances usually occurred in a mixture of dioxins (dioxin congeners in addition to TCDD).

Because TCDD is stored in fat tissue and has a long biologic half-life, internal exposure at generally constant concentrations may continue after episodic, high-level exposure to external sources has ceased. If a person had a high exposure, then large amounts of dioxins may be stored in fat tissue, which could be mobilized subsequently, as during weight loss. This would not be expected to be the case for non-lipophilic chemicals, such as cacodylic acid.

The mechanisms of possible effects on offspring differ greatly for men and women exposed to the COIs during their service in Vietnam. A father’s (paternal) contribution to adverse effects in his offspring is limited mainly to the contents of the fertilizing sperm and perhaps the seminal fluid (Lane et al., 2014). The sperm epigenome had long been believed to consist almost exclusively of a greatly condensed, transcriptionally inert haploid genome. As a result, it was thought that any paternally derived damage to the embryo or offspring would have to arise from changes in sequence (i.e., mutations) or arrangement of the sperm’s DNA. However, because dioxins are not genotoxic skepticism persisted concerning whether adverse outcomes in offspring could arise from paternal exposure to the COIs. Recent investigation of DOHaD and epigenetics in particular, however, have raised the possibility that epigenetic mechanisms might constitute a plausible mechanism by which parental exposures to the COIs might contribute to adverse outcomes in offspring.

Epigenetic effects are effects that elicit changes in gene expression without a change in DNA sequence but instead via covalent modifications to the DNA (usually involving methylation) or to other cellular components such as histones and miRNAs that interact with DNA to regulate gene expression and that persist through cell division (Jirtle and Skinner, 2007). Alterations in DNA expression arising from the epigenetic modification of an individual’s somatic cells may not be manifested for long periods of time. By definition, epigenetic transgenerational inheritance involves an alteration in the germ line that must be maintained for at least three generations following in utero exposures and for at least two generations after adult exposures (Jirtle and Skinner, 2007). The presumption is that this process requires exposure precisely at the time in germ line development when epigenetic programming is being established, although the mechanisms involved and whether they change from one generation to the next are not known (Skinner et al., 2010). (It should be noted that an adverse effect in the offspring of male or female Vietnam veterans need not be demonstrated to be transgenerational in

this strict sense in either humans or animals, but there must at least be coherent evidence of increased occurrence of the particular effect reported from epidemiologic studies of parents of the sex in question who were exposed to the COIs. Demonstration of epigenetic activity in animal experiments for the COIs would be regarded as supportive evidence of biologic plausibility.)

Paternally derived adverse outcomes in offspring associated with exposure to the COIs could be mediated by alterations of the DNA sequence, but, as noted in Chapter 4, genotoxic effects have not been shown for most of these chemicals. Thus, epigenetic modifications to the developing sperm epigenome, including altered RNAs, are a presumed mechanism (Krawetz, 2005). If the body burden were sufficiently high, then it is also possible that TCDD exposure might occur via absorption of seminal plasma through the vaginal wall, which could affect gestating offspring in an otherwise unexposed mother, although, as noted below, this scenario is unlikely.

A mother’s (maternal) contribution to a pregnancy and to her offspring is more extensive than the father’s contribution, and any damage to the resulting offspring or later generations can result from epigenetic changes in the egg or from direct effects of exposure on the fetus during gestation and on the neonate during lactation. Gestational exposures and resulting epigenetic change during development are consistent with the Barker hypothesis (also known as the developmental origins of disease), which proposes that some health outcomes occurring throughout the lifespan are established in part during fetal development. The Barker hypothesis also predicts a role for placental morphology and function in offspring health outcomes via epigenetic programming of the developing fetus (Barker and Thornburg, 2013), and TCDD has been reported to affect vascular remodeling of the placenta via an aryl hydrocarbon receptor (AHR)-dependent pathway (Wu et al., 2014). Herein, we review biologic plausibility and relevant data on female veterans and male veterans separately because the underlying pathways for adverse effects in offspring are so different.

Paternal Preconception and Postconception Exposure

There is particular interest in the possibility of paternally mediated effects on offspring and later generations because the vast majority of Vietnam veterans are male. Paternal exposures to TCDD or the other COIs could lead to developmental and later-life effects in offspring and potentially future generations by three feasible pathways. One involves direct alterations in the paternal fertilizing sperm cells that transmit adverse effects to resulting offspring through genetic or epigenetic mechanisms as delineated in Chapter 4. These effects would occur before conception. A second involves transmission of the contaminants to a female partner through seminal fluid during an established pregnancy, that is, after conception. A third is that TCDD contamination of the seminal fluid affects newly discovered functions.

Preconception Exposure

There is no evidence that dioxins can mutate DNA sequences; thus, genetic changes in sperm genes—as has been shown in connection with irradiation or the anticancer drug cyclophosphamide (Codrington et al., 2004)—due to preconception exposures to TCDD are not likely. On the other hand, the potential exists for TCDD to alter the sperm cells of adults before fertilization through epigenetic pathways. The sperm epigenome is distinctive from that of the egg (oocyte) or somatic cells (all other non-gamete cells in the body). The mature sperm cell has less global methylation than somatic cells, particularly at gene promoters, and unique DNA methylation marks (particularly on paternally imprinted genes) that put the sperm genomes in a pluripotent-like state before fertilization (Hales et al., 2011). However, rapid demethylation of most of the remainder of the paternal genome occurs shortly after fertilization (Dean, 2014), suggesting that additional changes are required for the nascent embryo to become truly pluripotent. Chemical alterations of DNA methylation foci of adult sperm have the potential to contribute to permanent effects in offspring, as suggested for male transmittance in fetal alcohol syndrome (Jenkins and Carrell, 2012a). During spermatogenesis in the adult, most sperm histones are replaced by protamines, which render the sperm transcriptionally quiescent and permit extensive DNA compaction. However, some core histones are retained in human sperm with appropriate epigenetic modifications to maintain open nucleosomes at sites that are important during embryo development (Casas and Vavouri, 2014), so their perturbation by exogenous chemicals remains a possibility. This is particularly important because although genome-wide DNA demethylation occurs in paternal DNA after fertilization (Dean, 2014) and should erase most sites that have been reprogrammed by chemicals, histone modification patterns are retained and thus may transmit chemical-induced alterations across generations (Puri et al., 2010).

Despite the exclusion of almost all cytoplasm, mature sperm have been found to carry a diverse spectrum of RNAs, including messenger RNAs (mRNAs), ribosomal RNAs (rRNAs), and small noncoding RNAs (miRNAs and piRNAs), which may affect the developing embryo (Casas and Vavouri, 2014; Hamatani, 2012; Kawano et al., 2012; Krawetz, 2005; Krawetz et al., 2011; Lane et al., 2014; Suh and Blelloch, 2011). For example, small RNAs of paternal origin may direct epigenetic modifications during embryo development and lead to changes in phenotype later in life (Hales et al., 2011). When newborn male mice were stressed by unpredictable separation from their mothers, miRNAs in their sperm have recently been shown to transmit the effects of this early trauma for two generations (Gapp et al., 2014). Heavy metals interact with sperm’s nuclear proteins, and this mechanism is suspected to be a basis of paternally mediated lead toxicity (Quintanilla-Vega et al., 2000). Disturbances in the establishment of the epigenetic marks in mature sperm may change cell fate in the early embryo and have effects throughout development and postnatal life (Jenkins and

Carrell, 2012b). Direct evidence of dioxin-mediated changes in the epigenome of mature sperm is not available. However, dioxins have been shown to modify DNA methylation in somatic cells (Hou et al., 2012), so an epigenetic pathway is biologically plausible. A related observation is a recent demonstration that the AHR plays an important role in normal sperm development (Hansen et al., 2014). To date, the only transgenerational effect shown in humans has been from a comparison of food supplies in Sweden during the 1800s and health outcomes in the children and grandchildren of men who were prepubescent when food supplies were relatively high or low. These studies found an association between high food supply levels in grandfathers with decreased longevity and increased risk of cardiovascular disease and diabetes in grandsons that was paternally transmitted, although no mechanistic information was obtained (Kaati et al., 2002, 2007). Whether transgenerational effects can occur in humans from chemical exposures is unknown at this time.

Postconception Exposure

Contaminants such as TCDD that are present in the tissues and blood of exposed males can be transported as parent compounds or metabolites into seminal fluid, the noncellular component of the ejaculate. Typically, concentrations of contaminants in seminal fluid are lower than those in serum, but direct assessments of the ratios of serum to seminal fluid in TCDD have not been reported. Seminal-fluid contaminants can be transmitted to a female during sexual intercourse and be absorbed through the vaginal wall; if the concentrations are high, then they could potentially affect a current pregnancy (Chapin et al., 2004; Klemmt and Scialli, 2005). TCDD and other persistent organic pollutants have been identified and quantified in the seminal plasma of exposed men, including Vietnam veterans (Schecter et al., 1996; Schlebusch et al., 1989; Stachel et al., 1989); thus, this transmission route is theoretically possible. In the Schecter study, serum TCDD was measured in 50 Vietnam veterans from Michigan who had a confirmed or self-reported potential for herbicide exposure and had blood drawn an average of 26 years after the possible exposure. Of those, six had TCDD greater than 20 parts per trillion (ppt) on a lipid-adjusted basis, which supports the idea that some veterans had high initial exposures. A subgroup of 17 men contributed semen at the time of blood draw, and dioxin congeners were analyzed in three randomly pooled samples—a process necessary to provide sufficient volume for chemical analysis. Although the measured concentrations were very low, the results documented the existence of dioxins and dibenzofurans in the seminal plasma of the veterans long after the possible herbicide exposure to TCDD-contaminated herbicides. Because results on serum and semen concentrations could not be linked to individual veterans and because it is unknown whether any of the ones who had high serum dioxin concentrations after 26 years contributed semen for the seminal-fluid measurements, the value of this information is slight.

Seminal-fluid concentrations of TCDD and related chemicals closer to the period of exposure in Vietnam have not been determined, so it is not possible to assess the clinical consequences of this exposure route for female partners and gestating offspring. Banked Ranch Hand specimens, however, might provide a valuable resource for comparing TCDD concentrations in serum and seminal fluid. A recent Institute of Medicine report describes available data and biospecimens from the Ranch Hand study and the potential for future analyses (IOM, 2015).

Despite the potential for a seminal fluid route of exposure, the critical question of dose sufficiency remains unanswered. That is, could absorbed TCDD concentrations be high enough to transmit adverse effects in the fetus? To answer that question, one must take into account several factors. First, the volume of seminal plasma is relatively low (1–5 mL) and because of leakage, only a fraction of seminal constituents is absorbed across the vaginal wall. Moreover, the dilution of absorbed chemicals in the female blood stream (i.e., in a high volume) before transmission across the placenta is estimated at 3 orders of magnitude or more (Klemmt and Scialli, 2005), which reduces a serum concentration of 20 ppt to a scale of parts per quadrillion (10^–15). Although studies to address the issue directly have not been undertaken, the dilution factor makes adverse fetal and offspring outcomes as a consequence of seminal plasma exposures to TCDD during pregnancy extremely unlikely. One caveat to this conclusion, however, is that seminal fluid is now known to play an important role in the metabolic phenotype of offspring because it stimulates embryotrophic factors (Bromfield, 2014; Bromfield et al., 2014). Whether TCDD contamination of the seminal fluid can affect this function is not known and should be tested.

Empirical Epidemiologic Evidence on Paternal Transmission

The idea that the exposure of either parent to a toxicant before conception could result in an adverse outcome in offspring is not new and remains a topic of much interest (Schmidt, 2013). Epidemiologic studies have reported occasional findings of paternally transmitted adverse outcomes associated with paternal exposures to certain agents, but none has been replicated convincingly. Even in instances in which an agent is recognized as mutagenic or potentially carcinogenic for exposed men, adverse consequences have not been demonstrated in their children. For example, the hypothesis was extensively investigated in the early 1990s in relation to fathers’ exposure to ionizing radiation before conception and an increase in leukemias in their offspring. The initial study (Gardner et al., 1990) was conducted in men who worked at the Sellafield nuclear facility in West Cumbria, United Kingdom. It was presumed that the men were exposed to radiation as a result of working at Sellafield. An association was found between radiation exposures to fathers before conception and an increase in leukemias among their children. However, later studies failed to confirm that finding (Draper et al., 1997; Kinlen, 1993; Kinlen et al., 1993; Parker et al., 1993; Urquhart et al.,

1991). Similarly, a rigorous follow-up of children of atomic-bomb survivors has not demonstrated increased risks of cancer or birth defects (Fujiwara et al., 2008; Izumi et al., 2003; Schull, 2003), and other studies of effects (birth defects and cancers) in the children of male cancer survivors after chemotherapy or radiation treatment have found little support for paternal transmission (Chow et al., 2009; Dohle, 2010; Howell and Shalet, 2005; Madanat-Harjuoja et al., 2010), although sperm and fertility clearly are adversely effected (Green et al., 2010).

An additional problem when trying to determine whether adult male exposure of any type (including to the COIs) can lead to pathological effects in descendants is that almost all experimental exposure studies to identify male transmission have been limited to developmental exposures in rodents (Guerrero-Bosagna and Skinner, 2014; Paoloni-Giacobino, 2014). An early experiment examining male mice treated with simulated Agent Orange mixtures prior to breeding with unexposed females failed to find an increase in a variety of different birth defects in progeny compared with the progeny of untreated males (Lamb et al., 1981). Epigenetic effects have been shown for male gametes in adult mice exposed to a relevant pesticide (methoxyclor) and fungicide (vinclozin) (Paoloni-Giacobino, 2014). However, the chemically induced DNA methylation changes in sperm DNA were not transmitted from one mouse generation to the next for imprinted genes; they were, presumably lost during the period of active demethylation that occurs shortly after fertilization. This observation suggests that transgenerational effects on imprinted genes in mice that might be paternally transmitted may not necessarily involve DNA methylation (Iqbal et al., 2015). Nonetheless, a recent study showed that odor fear conditioning in the father could be paternally transmitted to the F1 and F2 generations and implicated reduced DNA methylation in the responsible odor receptor gene (Dias and Ressler, 2013). Thus, more research is required to understand better how transgenerational effects can be transmitted paternally when they are demonstrated (Dias and Ressler, 2014).

The committee was unable to find a single instance of epidemiologic evidence that convincingly demonstrated paternal exposure to any particular chemical before conception resulting in cancers or birth defects in offspring. However, few data exist to address the hypothesis of paternal exposure and adverse effects in human offspring in which the exposure occurred before conception only to the father and was measured with an objective dosimeter. Thus, it is difficult to assert conclusively that the available epidemiologic evidence either supports or does not support paternal transmission; considerable uncertainty remains on many fronts and would presumably vary by agent and mode of exposure. Several systematic reviews of the topic have been conducted (Chia and Shi, 2002; Weselak et al., 2007, 2008; Wigle et al., 2007, 2008) and have not established firm relationships between specific agents and particular effects in offspring. Paternal occupation (by job title or job–exposure matrices) has been linked to an increased risk of selected birth defects (Desrosiers et al., 2012; Fear et al., 2007; Shaw et al., 2002) and neuroblastoma (De Roos et al., 2001a,b). Moreover, increased risks of

childhood brain cancer have been reported in relation to paternal exposure to selected pesticides, particularly herbicides and fungicides (van Wijngaarden et al., 2003), although the authors noted considerable uncertainty in the robustness of the findings. Therefore, the hypothesis that paternal preconception exposure to toxic agents may result in harm to their children remains unresolved in significant part because of the sparseness of epidemiologic research on the subject.

Maternal Exposure

A mother’s exposures can affect a pregnancy and the resulting offspring far more extensively than can paternal exposures. Because of the long half-life of TCDD and its bioaccumulation in adipose tissues, women exposed to herbicides in Vietnam would have the potential to expose their offspring to TCDD directly during later pregnancies. Thus, damage to the resulting offspring or future generations could result from epigenetic changes in an egg before conception or from the direct effects of exposure on the fetus during gestation and on the neonate during lactation. Dioxin in the mother’s bloodstream can cross the placenta and expose the developing embryo and fetus. Furthermore, the mobilization of dioxin during pregnancy or lactation may be increased because the body is drawing on fat stores to supply nutrients to the developing fetus or nursing infant. TCDD has been measured in circulating human maternal blood, cord blood, placenta, and breast milk (Suzuki et al., 2005), and it is estimated that an infant breastfed for 1 year accumulates a dose of TCDD that is six times as high as an infant not breastfed (Lorber and Phillips, 2002). Offspring effects of maternal exposures may not be manifested immediately and could be a result of dioxin-mediated reprogramming of developing organs and lead to a disease onset later in life. As noted above, placental structure and function are believed to play a major role in fetal programming, and TCDD has been shown to alter placental vascular remodeling (Wu et al., 2013, 2014).

As mentioned above in conjunction with the role of the placenta in fetal development, the developmental basis of adult disease (Barker et al., 2012) is being actively researched by investigating maternal nutritional exposures, stress, and alcohol exposure, and more recent studies have examined exposures to TCDD and other environmental toxicants. The molecular basis of the later-life effects is believed to be primarily epigenetic. Maladies that may be manifested later in life include neurologic and reproductive disorders, thyroid changes, diabetes, obesity, and adult-onset cancers. Furthermore, germ cells (eggs and spermatogonia) in offspring pass through critical developmental stages during fetal life (Hansen et al., 2014), and emerging evidence demonstrates that fetal exposures are capable of altering the germ cells epigenetically, resulting in a transmission of adverse effects to future generations (i.e., transgenerational inheritance) (Hansen et al., 2014).

Laboratory animal studies have established that TCDD can affect development, so a connection between TCDD exposure and effects on offspring,

including developmental disruption and disease onset in later life, is biologically plausible. It has been established in several animal studies that TCDD at high doses is a potent teratogen. Recent studies with rodent models have demonstrated male, female, and sex-independent effects in the immediate offspring of females exposed during pregnancy. These include epigenetic modification of imprinted genes (Somm et al., 2013), increased DNA methylation of the BRCA1 tumor suppressor gene in mammary tissue (Papoutsis et al., 2013), altered uterine response to estradiol (Burns et al., 2013), dysregulation of lipid metabolism in the presence of a high-caloric diet (Sugai et al., 2014), aberrant emotional behaviors (Nguyen et al., 2013), reduced capacity for lymphocyte differentiation (Ahrenhoerster et al., 2014), testicular inflammation (Bruner-Tran et al., 2014), and a variety of adult diseases including kidney, prostate, ovarian primordial follicle loss, and polycystic ovarian disease (Manikkam et al., 2012a). Transgenerational inheritance to the F3 generation was shown for the last two studies. However, definitive conclusions based on animal studies about the potential for TCDD to cause later-life toxicity in human offspring are complicated by differences in sensitivity and susceptibility among individual animals, strains, and species; by differences in route, dose, duration, and timing of exposure in experimental protocols and real-world exposure; and by differences in the toxicokinetics of TCDD between laboratory animals and humans. Experiments with 2,4-dichlorophenoxyacetic acid (2,4-D) and 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) indicate that they have subcellular effects that could constitute a biologically plausible mechanism for developmental effects, but only at very high doses. There is insufficient information on picloram and cacodylic acid to assess the biologic plausibility of their developmental or delayed effects in offspring.

Chapter 4 presents more detailed toxicologic findings that are relevant to the biologic plausibility of the outcomes discussed here.

BIRTH DEFECTS

A birth defect is an abnormality of structure, function, or metabolism, whether genetically determined or resulting from an environmental influence during embryonic or fetal life (Christianson et al., 2006). Other terms, often used interchangeably, are “congenital anomaly” and “congenital malformation.” Major birth defects, which occur in 2 to 3 percent of live births, are abnormalities present at birth that are severe enough to interfere with viability or physical well-being. Birth defects are detected in another 5 percent of babies through the first year of life. Genetic factors, exposure to some medications, exposure to environmental contaminants, occupational exposures, and lifestyle factors have been implicated in the etiology of birth defects (Christianson et al., 2006), although causes of the vast majority of birth defects are unknown. Most etiologic research has focused on the effects of maternal and fetal exposures, but as discussed in the beginning of this chapter, it is theoretically possible that epigenetic alterations of

the paternal gamete caused by preconception exposures could result in paternally mediated effects. It should be noted that a substantial amount of epidemiologic research on suspect toxic agents has been conducted, but none of it has not definitively established paternal preconception exposures as a contributing factor to the occurrence of birth defects (Chow et al., 2009; Desrosiers et al., 2012; Dohle, 2010; Schull, 2003).

Conclusions from VAO and Previous Updates

The committee responsible for VAO concluded that there was inadequate or insufficient evidence to determine whether there is an association between exposure to 2,4-D, 2,4,5-T or its contaminant TCDD, picloram, or cacodylic acid and birth defects in offspring. Additional information from the Air Force Health Study (AFHS) available to the committee responsible for Update 1996 led it to conclude that there was limited or suggestive evidence of an association between at least one of the COIs and spina bifida in the children of veterans; there was no change in the conclusions regarding other birth defects. The committee for Update 2002, which reviewed the study of female Vietnam veterans that reported significant increases in birth defects in their offspring (Kang et al., 2000a), did not find those results adequate to modify prior conclusions. Nonetheless, Congress did mandate that a number of birth defects in the children of female Vietnam veterans be assigned service-related status. Later VAO committees have not encountered enough additional data to merit changing the conclusion that the evidence is inadequate to support an association between exposure to the COIs and birth defects (aside from spina bifida) in the offspring of either male or female veterans. Summaries of the results of studies of birth defects and specifically of neural-tube defects that were reviewed in the current report and in earlier VAO reports are in Tables 10-1 and 10-2, respectively.

Update of the Epidemiologic Literature

No Vietnam-veteran, occupational, or environmental studies of exposure to the COIs and LBW or PTD have been published since Update 2012.

Case-Control Studies

Since Update 2012, two studies have examined residential proximity to applications of commercial pesticides in California and birth defects. Using data from the California Pesticide and Birth Defects Monitoring programs, Carmichael et al. (2013) examined pesticide applications within 500 meters of a residence and hypospadias in 690 cases and 2,195 controls. Among the cases classified as the least severe, applications of 2,4-D within 500 meters of the home occurred during the first 14 weeks of pregnancy (the critical window of exposure for birth

TABLE 10-1 Selected Epidemiologic Studies—Birth Defects in Offspring of Subjects^a (Shaded entries are new information for this update)

NOTE: 2,4-D, 2,4-dichlorophenoxyacetic acid; 2,4,5-T, 2,4,5-trichlorophenoxyacetic acid; AFHS, Air Force Health Study; BWIS, Baltimore–Washington Infant Study; CDC, Centers for Disease Control and Prevention; CI, confidence interval; COI, chemical of interest; EOI, Exposure Opportunity Index; JEM, job–exposure matrix; IARC, International Agency for Research on Cancer; MCPA, 4-chloro-2-methylphenoxyacetic acid; MIH, molar incisor hypomineralization; NIOSH, National Institute for Occupational Safety and Health; nr, not reported; ns, not significant; OFFHS, Ontario Farm Family Health Study; OR, odds ratio; PCB, polychlorinated biphenyl; PCDD, polychlorinated dibenzodioxins; PCDF, polychlorinated dibenzofurans; SEA, Southeast Asia; TEQ, (total) toxic equivalent; TCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxin; VA, US Department of Veterans Affairs.

^aUnless otherwise indicated, studies show paternal exposure.

^bGiven when available; results other than estimated risk explained individually.

defects) for five cases. After adjusting for confounders, the observed risk was elevated (odds ratio [OR] = 2.11, 95% confidence interval [CI] 0.80–5.56) but statistically imprecise due to the small number of exposed cases. No increased risk was observed for cases classified as more severe and applications of 2,4-D.

Using a similar strategy for the California cases and controls enrolled in the US National Birth Defects Prevention Study (NBDPS), a population-based case-control study of congenital malformations, Carmichael et al. (2014), Yang et al. (2014) and Shaw et al. (2014) examined a number of birth defects that were verified by clinical geneticists. Cases and controls were considered exposed when applications of 2,4-D occurred within 500 meters of the maternal residence during the critical period of pregnancy. After an adjustment for confounders, elevated risks of pulmonary valve stenosis (OR = 2.9, 95% CI 1.0–7.9), atrial septal defect (OR = 2.3, 95% CI 1.2–4.5), and anencephaly (OR = 2.0, 95% CI 0.8–5.1) were observed. No association was observed for cleft lip with or without cleft palate, and there were insufficient numbers of exposed cleft palate (n = 4) and spina bifida (n = 4) cases to calculate adjusted risks. Finally, applications of 2,4-D were associated with an increased risk of gastroschisis (OR = 1.6, 95% CI 0.8–3.2).

TABLE 10-2 Selected Epidemiologic Studies—Neural-Tube Defects in Offspring of Subjects^a (Shaded entries are new information for this update)

NOTE: 2,4,5-T, 2,4,5-trichlorophenoxyacetic acid; CDC, Centers for Disease Control and Prevention; CI, confidence interval; COI, chemical of interest; nr, not reported; SEA, Southeast Asia; TCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxin; VES, Vietnam Experience Study.

^aUnless otherwise indicated, studies show paternal exposure.

^bGiven when available; results other than estimated risk explained individually.

^cOf four neural-tube defects reported in Ranch Hand offspring, two were spina bifida (high dioxin exposure), one spina bifida (low dioxin), one anencephaly (low dioxin); no neural-tube defects reported in comparison cohort; 454 postservice births studied in Ranch Hand veterans; 570 in comparison cohort.

^dGreenhouse workers would not have been exposed to the COIs.

^eCalculated from data presented in the paper.

An additional study examined musculoskeletal defects and maternal occupational exposure to pesticides in the NBDPS, but exposure classification was limited to herbicides and did not meet the exposure classification criteria for the COI (Kielb et al., 2014).

Biologic Plausibility

2,4-D has been previously shown to be a teratogen, although at exposures that exceed maternal renal clearance and thus are not relevant to herbicide exposure in Vietnam. A recent study has shown for the first time that late in utero and early postnatal 2,4-D exposure can result in nephrotoxicity in offspring, although at one-sixth of the LD₅₀ (Troudi et al., 2011). Other herbicides of interest can induce fetal malformations but typically only at high doses that are toxic to pregnant women. TCDD is a potent teratogen in all laboratory species that have been studied, although the patterns of birth defects that are produced are often species specific. However, specific mechanisms that link TCDD exposure to specific birth defects have not been fully elucidated.

A variety of animal model studies, including in utero exposures, work with cultured cells, and zebrafish embryos, have investigated the mechanisms underlying various TCDD-induced birth defects including hydronephrosis, cleft palate, reproductive organ anomalies, neurogenesis, and perturbed heart, kidney, and lung development (Dong et al., 2010; Falahatpisheh et al., 2011; Jacobs et al., 2011; Lanham et al., 2012; Latchney et al., 2011; Neri et al., 2011; Tait et al., 2011; Yamada et al., 2014; Yoshioka et al., 2012; Yuan et al., 2012). Interestingly, the AHR is required for TCDD-induced birth defects. In contrast, the induction of cytochrome P4501A1 is not required (Dragin et al., 2006; Jang et al., 2007; Mimura et al., 1997). When pregnant AHR-null mice are exposed to TCDD, the fetuses do not exhibit any of the typical developmental malformations associated with TCDD exposure, but fetuses of TCDD-exposed pregnant CYP1A1-null mice do. In addition, an AHR antagonist can attenuate TCDD-induced birth defects in mice. Thus, the activation of the AHR by TCDD during development appears to be a key first step in mediating TCDD’s developmental toxicity, but this step does not depend on CYP1A1 activity. Although structural differences in the AHR have been identified among species, it functions similarly in animals and humans. Therefore, a common mechanism mediated by the AHR in which tissue growth and differentiation processes are affected probably underlies the developmental toxicity of TCDD in humans and animals.

Antioxidant treatment provides protection against some TCDD-induced teratogenicity, which suggests that reactive oxygen species might be involved in the pathways that lead to these structural changes (Jang et al., 2008). A few studies indicate the stem cells and organ-specific progenitor cells may be direct targets and that maternal TCDD exposures interfere with proliferation and cell differentiation through the AHR and result in defects in organ morphogenesis (Latchney et al., 2011; Neri et al., 2011). Few laboratory studies of potential male-mediated developmental toxicity (and specifically birth defects) attributable to exposure to TCDD and herbicides have been conducted. As noted, the feeding of simulated Agent Orange mixtures to male mice produced no adverse effects in offspring (Lamb et al., 1981).

In sum, studies with maternal exposure in animal models suggest that a role for TCDD and related chemicals in causing birth defects is plausible and also that the AHR plays a causal role. However, translating these results to human populations has been difficult.

Synthesis

Embryonic and fetal development in rodents is sensitive to the toxic effects of exposure to TCDD and dioxin-like chemicals. It is clear that the fetal rodent is more sensitive to the adverse effects of TCDD than the adult rodent. Human data are generally lacking, however, and the sensitivity to developmental disruption in humans is less apparent, in part because contemporary studies of environmental

dioxin exposure and birth defects have involved extremely low exposures. As noted, recent human population-based studies have provided mixed results in attempts to link TCDD or the other COI exposures to birth defects. Two studies in California found no or very limited evidence for associations between population exposures and neural tube defects, orofacial clefts, gastroschisis, and congenital heart defects (Carmichael et al., 2014; Shaw et al., 2014; Yang et al., 2014). Congenital heart defects are the most common congenital malformations and persistent organic pollutants (POPs) are suspected of playing a contributing role, but firm statistical links are still lacking (Gorini et al., 2014). An additional California study looked for a link between pesticide exposures and hypospadias, but also failed to make a strong association (Carmichael et al., 2013). The studies since Update 2012 that have assessed exposure to relevant chemicals and congenital malformations examined only maternal or residential exposure, which is of little relevance to the majority of Vietnam veterans. Furthermore, those case-control studies were conducted in populations exposed to contemporary concentrations, which may be too low for adverse fetal effects to be observed. The studies were well designed and adjusted for important confounders, but they do not provide evidence of an association at these exposure levels.

Given the long-standing concern of the Vietnam veterans about the potential of the COIs to adversely affect the health of their children, birth defects and childhood cancers have been among the outcomes considered by VAO committees since the first comprehensive review published in 1994.

As indicated in the section above summarizing the findings of prior VAO committees, the committee for the second VAO report (Update 1996) concluded that there was “limited or suggestive” evidence of an association between herbicide exposure and a single type of birth defect (the neural-tube defect spina bifida). That committee extracted an item of evidence concerning spina bifida from a new publication regarding the AFHS (Wolfe et al., 1995). The authors of the paper noted that the category of nervous system birth defects involved too few case to permit analysis (three from the comparison group; in the categories for serum dioxin levels measured in the fathers who served in Operation Ranch Hand [ORH], zero with background levels, two with low levels, and three with high levels). The committee for Update 1996 calculated a marginally significant exact p-value of 0.04 for the three cases of spina bifida and one case of anencephaly among the children of the ORH subjects in comparison to zero cases for the fathers in the comparison group (the nature of the remaining nervous system defects—three in the controls and one in the high serum TCDD group of ORH veterans—is not evident in the original paper). This result was added to a fairly extensive body of evidence regarding birth defects overall that the preceding committee for VAO had judged to be imprecise and inconsistent and to contain little evidence of an association with paternal occupational exposure to herbicides or dioxin. As shown in Table 10-2, that dataset contained two studies (CDC, 1989a; Erickson et al., 1984a,b) with results on neural-tube defects consistent with an

association with paternal exposure to the COIs. Because the Agent Orange Act did not have provisions for compensation of the veterans’ offspring, Congress passed legislation to permit this.

Although a number of studies published since Update 1996 have examined exposures to pesticides and spina bifida, the four that examined paternal exposure and spina bifida (Blatter et al., 1997; Dimich-Ward et al., 1996; Garry et al., 1996; Kristensen et al., 1997) used paternal occupation (e.g., farmer, pesticide applier) as the basis for exposure classification and were not able to examine the COIs specifically. The remaining studies published since the 1996 report examined residential proximity to pesticide applications. As previously discussed, this form of exposure classification assumes the potential for both maternal and paternal exposure prior to and during pregnancy but cannot verify that such an exposure occurred.

Only a very small portion of Vietnam veterans are women, but a VA study (Kang et al., 2000a) of their health and reproductive history in comparison to their non-deployed Vietnam-era counterparts found a significant increase in birth defects overall. The committee for Update 2002 reviewed this study but had reservations about exposure being defined simply as deployment and also about the fact that verification with medical records of the problems these women reported in their children was less complete than planned. Congress did, however, legislate eligibility for compensation for children of female Vietnam veterans with a broad range of birth defects not attributable to familial conditions.

It is extremely difficult to conduct epidemiology studies assessing risks to children arising from their parents’ exposure, particularly when a distinction between maternal and paternal contributions is sought. As with studies of paternal exposure, additional confirmatory epidemiologic evidence has not become available to support an association of spina bifida with maternal exposure to the components of the herbicides sprayed in Vietnam. In fact, an increase in birth defects following adult exposure of only the male parent has not (as yet) been definitively demonstrated for any toxic agent.

With the continued increase in the number of women serving during deployments, concern about adverse consequences in the offspring of veterans merits increased attention. For Vietnam veterans, however, the existing evidence supporting an increase in spina bifida specifically in the children of men or women who were deployed is very sparse.

Conclusions

The committee concludes that the new evidence concerning the occurrence of birth defects in association with exposure to the COIs, in combination with existing evidence, remains inadequate and insufficient to support an association for birth defects overall in the children of Vietnam veterans. In light of the fact that evidence anticipated by the committee for Update 1996 that would support an association between spina bifida and paternal exposure to the COIs has not

materialized from the AFHS or from any other population with relevant exposures, the committee concludes that spina bifida should be demoted from the category of limited or suggestive evidence of an association to the default category of inadequate or insufficient evidence of an association. Increased scrutiny of mechanisms by which paternal exposure might contribute to adverse effects in offspring has not as yet definitively established the biologic plausibility of this phenomenon, whereas understanding of how maternal exposures may disrupt fetal development has grown substantially. There are, however, no epidemiologic results supporting an association between maternal exposure to the COIs and spina bifida specifically, so spina bifida in association with exposure of either parent has been moved to the inadequate and insufficient category of association.

CANCERS IN OFFSPRING

The American Cancer Society (ACS) estimated that 11,630 children under 15 years old will receive a diagnosis of cancer in the United States in 2013 (ACS, 2013a). The treatment and supportive care of children who have cancer have continued to improve. The 5-year survival rate for children who receive a cancer diagnosis has increased from less than 60 percent in the 1970s to more than 80 percent in 2013. Despite those advances, cancers remain the leading cause of death from disease in children under 15 years old, and 1,310 deaths were projected for 2013 (ACS, 2013a).

Leukemias are the most common cancer in children, accounting for about one-third of all childhood cancer cases. In 2015, ACS forecast that about 3,314 children would receive a leukemia diagnosis (ACS, 2015). Of those, nearly 2,500 would have acute lymphocytic leukemia (ALL), and most of the rest would have acute myeloid leukemia (AML). AML (International Classification of Diseases, Revision, 9th Revision [ICD-9] 205) is also referred to as acute myelogenous leukemia or acute nonlymphocytic leukemia. For consistency, this report uses “acute myeloid leukemia,” or AML, regardless of the usage in the source materials. ALL is most common in early childhood, peaking at the ages of 2–3 years, and AML is most common during the first 2 years of life. ALL incidence is consistently higher in boys than in girls; AML incidence is similar in boys and girls (NCI, 2001). Through early adulthood, ALL rates are about twice as high in whites as in blacks; AML exhibits no consistent pattern in this respect. Chapter 8 contains additional information on leukemias as part of the discussion of adult cancers.

The second-most common group of cancers in children consists of cancers of the central nervous system—the brain and the spinal cord. Other cancers in children include lymphomas, bone cancers, soft-tissue sarcomas, renal cancers, eye cancers, and adrenal cancers. In contrast with adult cancers, relatively little is known about the etiology of most childhood cancers, especially about potential environmental risk factors and the effects of parental exposures.

Conclusions from VAO and Previous Updates

The committee responsible for VAO concluded that there was inadequate or insufficient evidence to determine whether there is an association between exposure to 2,4-D, 2,4,5-T, TCDD, picloram, or cacodylic acid and childhood cancers. The additional information available to the committees responsible for Update 1996 and Update 1998 did not change that conclusion. The committee responsible for Update 2000 reviewed the material in earlier VAO reports and in newly available published literature and concluded that there was limited or suggestive evidence of an association between exposure to at least one of the COIs and AML. After the release of Update 2000, investigators involved in one study discovered an error in their published data. The Update 2000 committee reconvened to evaluate the previously reviewed and new literature regarding AML, and it produced Acute Myelogenous Leukemia (IOM, 2002). It reclassified AML from “limited/suggestive evidence of an association” to “inadequate evidence to determine whether an association exists.”

Table 10-3 summarizes the results of the relevant studies. The committees responsible for Update 2002, Update 2004, Update 2006, Update 2008, Update 2010, and Update 2012 reviewed the material in earlier VAO reports and in newly available published literature and agreed that there remained inadequate or insufficient evidence to determine whether there is an association between exposure to the COIs and childhood cancers.

Update of Epidemiologic Literature

No Vietnam-veteran, occupational, or environmental studies of exposure to the COIs and childhood cancers have been published since Update 2012.

Case-Control Studies

Glass et al. (2012) examined ALL and parental occupational exposure to pesticides around the time of conception in a case-control study. Job–exposure modules were used to collect parental occupational history, and an expert rater classified pesticide exposure. A strength of this study was the ability to examine both maternal and paternal exposures. After adjustment for confounders among the 327 cases with paternal exposure information, 8 reported exposure to phenoxy herbicides. Only 2 of the 378 cases with maternal information reported exposure to phenoxy herbicides. No associations were observed with ALL and occupational exposure to phenoxy herbicides.

Metayer et al. (2013) examined 252 ALL cases and 308 controls in the Northern California Childhood Leukemia Study, a population-based case-control study. Pesticides, including 2,4-D, were detected in the majority of house dust samples collected in this study as a proxy for exposure during pregnancy. However, no

TABLE 10-3 Selected Epidemiologic Studies—Childhood Cancer^a (Shaded entries are new information for this update)

NOTE: 2,4-D, 2,4-dichlorophenoxyacetic acid; AHS, Agricultural Health Study; AIHW, Australian Institute for Health and Welfare; ALL, acute lymphocytic leukemia; AML, acute myeloid leukemia; CATI, computer-assisted telephone interviewing; CDC, Centers for Disease Control and Prevention; CI, confidence interval; COI, chemical of interest; GCT, germ-cell tumor; HL, Hodgkin lymphoma; ICD, International Classification of Diseases; NHL, non-Hodgkin lymphoma; nr, not reported; TCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxin; VES, Vietnam Experience Study.

^aUnless otherwise indicated, studies show paternal exposure.

^bGiven when available; results other than estimated risk explained individually.

^cOf the 12, nine were observed, three additional cases estimated to have occurred in portion of cohort whose data were not validated.

difference in ALL risk was observed between cases and controls (OR = 0.96, 95% CI 0.85–1.08).

Biologic Plausibility

Paternal or maternal exposure to xenobiotics potentially could increase the susceptibility of offspring to cancer through multiple mechanisms. Susceptibility could be increased by causing a tumor-promoting mutation in germ cells that would be present in all of the somatic cells of the child. This de novo mutation could then be passed on to subsequent generations via Mendelian inheritance, assuming that the child survived to reproduce. However, as discussed earlier in this chapter and in earlier chapters, TCDD and other COIs are not genotoxic (i.e., they do not cause mutations), which makes the mutation-induction and inheritance

scenario unlikely. Alternatively, a maternally mediated increase in susceptibility to childhood cancers could result from direct exposure of a fetus in utero or the newborn via lactation to a xenobiotic that induces epigenetic alterations that increase cancer susceptibility.

The biological plausibility overview for this chapter presented several pre- and post-conception scenarios for how toxicant exposures could cause disease in first-generation offspring and perhaps in later generations based on epigenetic mechanisms (Vaiserman, 2014). Perhaps the most straightforward scenario is in utero exposure affecting the developing epigenome, which predisposes the child to cancer. The best example of this happening is when otherwise very rare vaginal cancers arose in the daughters of women who took the estrogenic agent diethylstilbestrol (DES) to prevent miscarriage (Herbst et al., 1971). Thus this scenario is quite plausible for humans. Although TCDD is an antiestrogen, as noted, its toxicity via the AHR likely involves transcriptional changes that could induce epigenetic mechanisms. With regard to cancers, if the affected gene or genes are involved in cancer pathways and epigenetic modifications stabilize the gene-expression changes, then the susceptibility to cancer could increase.

Prenatal TCDD exposure of rats is associated with altered mammary gland differentiation and an increase in the number of mammary adenocarcinomas (Brown et al., 1998). Perhaps related, prenatal TCDD exposure led to increased DNA methylation at the BRCA1 (breast cancer) gene promoter in the female offspring of exposed pregnant rats (Papoutsis et al., 2013). The demonstration that early postnatal TCDD exposure does not increase mammary-cancer risk (Desaulniers et al., 2004) does not contradict the finding that TCDD-induced changes in utero mediate the increase in cancer susceptibility (Fenton et al., 2000, 2002), and is consistent with ultimate carcinogenic effect being greatest when epigenomic changes are most dynamic. Thus, developmental epigenetic alterations may be involved in the prenatal effects. TCDD has been shown to suppress the expression of two tumor-suppressor genes, p16^Ink4a and p53, via an epigenetic mechanism that appears to involve DNA methylation (Ray and Swanson, 2004). Similarly, it was reported that prenatal TCDD exposure increases methylation of two growth-related imprinted genes, H19 and Igf2, in the developing fetus (Wu et al., 2004).

No direct evidence from animal models shows that TCDD increases the risk of childhood cancers, such as acute leukemia and germ-cell tumors, although a recent study showed a reduced capacity of hematopoietic stem cells to undergo differentiation in offspring (Ahrenhoerster et al., 2014). Emerging research suggests that prenatal TCDD exposure can disrupt epigenetic imprinting patterns and alter organ differentiation and thus could contribute to an increased susceptibility to cancer later in life. Smith et al. (2005) showed that chromosomal rearrangements associated with childhood ALL are evident in the neonatal blood spots, which suggests that childhood leukemias begin before birth, perhaps due to maternal exposures to carcinogenic xenobiotics.

Synthesis

No associations were observed in the two case-control studies that considered childhood ALL and exposure to phenoxy herbicides and to 2,4-D in particular. Furthermore, evidence is sparse that exposure to the COIs increases the risk for childhood cancers.

Conclusions

On the basis of the evidence reviewed here and in previous VAO reports, the committee concludes that there is inadequate or insufficient evidence to determine whether there is an association between exposure to the COIs and childhood cancers.

EFFECTS OCCURRING LATER IN OFFSPRING’S
LIFE OR IN LATER GENERATIONS

In response to a special request from the Department of Veterans Affairs, continuing inquiries from veterans and their families, and increasing attention in research efforts, the committee for Update 2010 addressed whether it was feasible to assess associations between exposure to the herbicides sprayed in Vietnam and health effects that occur later in the lives of children of Vietnam veterans and even in their grandchildren; such associations had not been formally reviewed in prior VAO updates. The previously considered outcomes of birth defects observable within the first year of life and childhood cancers (diagnosis before the age of 18 years) were augmented to include all cancers and physical and neurobehavioral problems that might be manifested at any age, thereby broadening the scope of DOHaD research relevant to the VAO mission.

In addition, for the first time, the committee for Update 2010 explored the possibility of transgenerational effects resulting from exposure-related epigenetic changes in the parents or exposed fetuses that would lead to adverse health effects in later generations, such as grandchildren. As noted above, effects in persons exposed in utero are not considered transgenerational because the fetus was likely exposed directly. This exception includes the children of women exposed in Vietnam even if they are conceived after their tour of duty was over because TCDD remains in the body for a long time and is mobilized during pregnancy. Likewise, the children of men exposed to TCDD in Vietnam and born after the soldiers’ tour of duty was over could possibly have health outcomes due in part to TCDD’s effect on the sperm epigenome. In contrast, any adverse health effects in grandchildren associated with exposure would be considered to be transgenerational.

Conclusions from VAO and Previous Updates

The potential effect that herbicide exposure in male and female Vietnam veterans would have on the development of diseases other than cancers in the veterans’ children after the first year of life or in later generations had not been considered in updates before Update 2010.

For Update 2010, epidemiologic studies that evaluated the potential for effects of maternal or paternal exposure to the COIs in offspring were identified. Rather than identifying specific diseases in offspring, much of the research involved the measurement of physiologic biomarkers that might indicate a potential for disease development later in life. The committee for Update 2010 therefore cautioned strongly that the clinical consequences of any observed changes are highly uncertain. The committee maintained its standard requirement for exposure specific to components of the herbicides sprayed in Vietnam.

Although, as noted above, it may be physiologically possible for paternal exposure to cause changes in offspring that are manifested later in life, none of the published epidemiologic studies assessed such potential. Thus, the observation of any changes reported in studies discussed in this section should be applicable only to children born to female Vietnam veterans during or after their deployment in Vietnam. Thus, no transgenerational studies have been reported to date.

Changes Detected in Children After Parental Exposure

Growth and Development

Since Update 2012, three studies have examined exposure to environmental contaminants, including PCBs, and the subsequent growth and development of children. Delvaux et al. (2014) measured the height and weight of 114 children included in the Flemish Environment and Health Study when they were between the age of 7 and 9 years old. Dioxin-like activity was measured in the cord blood collected at birth with the CALUX assay. No association between prenatal exposure to dioxin-like chemicals and height or weight was observed 7 to 9 years after birth.

In another cohort, Wohlfahrt-Veje et al. (2014) measured dioxin levels and PCBs in maternal breast milk and markers of growth and development at a number of time points from birth through 36 months of age in 418 mother–child pairs. For the 368 pairs with non-smoking mothers, in the first month after birth total TEQs were associated with a depression in adjusted measures of weight (−0.38, 95% CI −0.76–0.00), length (−0.34, 95% CI −0.70–0.01), and fat percentage (−0.52, 95% CI 1.08 to −0.03). However, the changes observed between 0 and 18 months in weight (0.69, 95% CI 0.18–1.21) and height (0.79, 95% CI 0.33–1.26)

indicated accelerated growth. With adjustment for maternal smoking during pregnancy, similar findings were observed for all 418 pairs.

Sioen et al. (2013) enrolled 270 mothers and newborns in a prospective cohort study of behavior and markers of behavior and neurodevelopment. Dioxin-like activity was measured in cord blood using the CALUX assay. Prenatal exposure to dioxin like compounds was not associated with assessed behavior in the children at age 7–8.

Cognitive or Motor Development

In a study from Vietnam, Tai et al. (2013) enrolled 216 infant–mother pairs and assessed markers of neurodevelopment through 4 months of age. Dioxin levels were measured in breast milk, and infant neurodevelopment was measured using the Bayley Scales of Infant and Toddler Development. After adjustment for confounders (gender, parity, gestational week, age, birth weight, education, age, socioeconomic status, alcohol consumption, smoking status, environmental tobacco smoke, and maternal residence), PCDDs/Fs ≥ 17.6 pgTEQ/g lipid were significantly correlated with lower cognitive function and fine motor skills when compared to PCDDs/Fs ≤ 7.4 pq TEQ/g lipid (p = 0.009 and 0.030, respectively). Similarly, infants with daily dioxin intake (≥ 118.2 pg TEQ/kg/day) from breast milk had lower mean measures of cognitive function and fine motor skills compared to infants with ≤ 49.8 pg TEQ/kg/day (p = 0.006 and 0.017, respectively).

In 2014 ten Tusscher et al. published a study that compared dioxin levels in breast milk with subsequent neurodevelopment between 7 and 12 years of age for 41 children and for 33 of those same children between 14 and 18 years of age. Psychologists, parents, and teachers completed standardized scales designed to measure neurodevelopmental outcomes and behaviors in the children (the Weschler Intelligence Scale for Children [WISC], the Child Behavior Checklist and the Teacher Report Form, respectively). Increased social problems (β = 0.03, p = 0.001), aggressive behavior (p = 0.001), and problems thinking (p = 0.005) as measured by the Teacher Report Form were associated with increasing postnatal dioxin levels. In addition, prenatal exposure was associated with a number of behavioral indicators measured on the Child Behavior Checklist, including social problems (p = 0.001), anxiety (p = 0.002), and internalized behavior (p = 0.007). In contrast, behavior assessed by a psychologist using the WISC-R showed no associations with prenatal or postnatal exposure to dioxin.

Winneke et al. (2014) examined hormonal influences on behavioral development in 232 mother–child pairs using the Preschool Activities Inventory to measure markers of behavior that may indicate potential sexual dimorphism. Dioxin was measured in both maternal blood collected in the period 28–42 weeks after conception and breast-milk samples. While most comparisons were not statistically significant, a doubling of maternal blood levels of PCDD/F and PCDD/F+PCB

combined was associated with a difference in the score for markers of sexual dimorphism in boys (β = 2.60, 95% CI 0.39–4.80), but not in girls (β = 2.62, 95% CI 0.52–4.71). Similarly PCDD/F and PCDD/F+PCBs measured in breast milk were associated with the same markers in boys (β = 4.02, 95% CI 1.77–6.28; β = 3.90, 95% CI 1.74–6.06, respectively). Prenatal measures of dioxin were not associated with behavioral markers in girls. However, PCDD/F+PCBs as measured in breast milk was associated with markers of dimorphism in girls (β = −4.22, 95% CI −7.24 to −1.19).

Immune-Cell Populations and Prevalence of
Allergies or Asthma in Children

Hansen et al. (2014) measured prenatal exposures to POPs and the development of asthma in 965 children enrolled in the Danish Fetal Origins cohort study. Six PCB congeners were measured from maternal serum collected during pregnancy and grouped by dioxin-like activity. However TEQs were not calculated. The confirmation of prescriptions for asthma medication data, as obtained from the medication registry, was used to classify asthma cases for children ranging in age from 6 to 20 years. Asthma risk was increased (RR = 1.90, 95% CI 1.12–3.23) for children whose mothers had the highest tertile of PCB 118 exposure during pregnancy (−20.0–0.61 ng/ml). Similarly, risk was increased (RR = 1.75, 95% CI 1.02–2.98) for children whose mothers were classified in the highest tertile of exposure for dioxin-like PCBs (> 0.96 pmol/ml–4.10 pmol/ml).

Offspring Reproductive Function

In addition to evaluating the overall health and survival of the children of Vietnam-era veterans, The Australian Vietnam Veterans Family Study (ADVA, 2014b) assessed a number of self-reported outcomes related to the reproductive success of the children of these veterans. In comparing self-reports from children of deployed veterans to those of children of non-deployed veterans, no significant differences were found with respect to difficulty in conceiving or in the incidence of miscarriages, stillbirths, or the specific birth defects spina bifida and cleft lip or palate.

Two studies on subcohorts of the collaborative European NewGeneris study of mother–child pairs examined anogenital distance and maternal exposure to dioxin and dioxin-like compounds. Anogenital distance has historically been used as a marker of androgen function in toxicology studies involving animals. Recently, this marker has been used in epidemiology studies to examine the effects of exposures that may affect hormonally related outcomes. Using the Greek and Spanish subcohorts, Papadopoulou et al. (2013b) examined anogenital distance in light of self-reported information on maternal diet during pregnancy. A high-fat diet was correlated with dioxin-like activity as measured in 121 maternal blood

samples in this cohort. High fat-in-diet scores for the mothers were associated with a decrease in anoscrotal distance in their infant sons (β = −4.2, 95% CI –6.6 to −1.8). Dietary fat intake was not associated with anoforchetal distance in the newborn females or anogenital distance in boys or girls at 1–2 years of age.

Vafeiadi et al. (2013) used dioxin-like activity measured in maternal blood samples collected at the time of delivery to study the same outcome. Included in the analyses were 237 newborns (119 boys and 118 girls) and 462 children aged 1–31 months (239 boys and 223 girls) from the same two subcohorts of the NewGeneris study. No associations between dioxin-like activity and three measures of anogenital distance were observed in either female newborns or girls. In male newborns, dioxin-like activity was significantly associated with shortened anogenital distance (β = 0.44 mm, 95% CI –0.80 to –0.08) per a 10 pg CALUX-toxic equivalent/g lipid. A similar association, although not statistically significant, was observed in boys. These two sets of findings about anogenital distance are consistent, but this use of the actual measures of dioxin-like activity would be considered more reliable than the indirect measure of gestational exposure based on maternal diet used by Papadopoulou et al. (2013b).

Biologic Plausibility

Results of studies in rodent models provide support for the idea that prenatal exposure to TCDD can result in adverse effects in offspring later in life, including immune disorders, behavioral disturbances, reproductive impairment, kidney disease, and cancers (Foster et al., 2010; Prescott, 2011; Puga, 2011; Takeda et al., 2012). Using two mouse models, investigators showed that prenatal TCDD (2.5–5.0 mg/kg) modified multiple immune signatures in adult offspring that were indicative of adult-onset autoimmunity (Holladay et al., 2011). Adult-onset inflammatory disease and lupus-like autoimmunity were also observed in mice at 36 weeks of age after high-dose prenatal TCDD exposures (Mustafa et al., 2011). A single prenatal exposure of rats to TCDD (0.7 mg/kg of body weight) reduced brain developmental myelination and compromised remyelination potential in adults (Fernández et al., 2010), and in utero TCDD in mice altered neural progenitor differentiation (Mitsuhashi et al., 2010). However, another study suggested that, unlike murine neurospheres (which represent neural progenitor cells), human neurospheres were nonresponsive to TCDD because of lack of the AHR—an indication of species specificity in response (Gassmann et al., 2010). Perinatal TCDD (0.2–0.4 mg/kg of body weight) in rats perturbed neuroendocrine function as measured by thyrotropin and growth hormone concentrations in exposed offspring through peripubertal postnatal day 30, which supports the idea of continued later-life thyroid hormone disturbances (Ahmed, 2011). These include the epigenetic modification of imprinted genes (Somm et al., 2013), increased DNA methylation of the BRCA1 tumor suppressor gene in mammary tissue (Papoutsis et al., 2013), altered uterine response to estradiol (Burns et al., 2013),

dysregulation of lipid metabolism in the presence of a high-caloric diet (Sugai et al., 2014), aberrant emotional behaviors (Nguyen et al., 2013), a reduced capacity for lymphocyte differentiation (Ahrenhoerster et al., 2014), testicular inflammation (Bruner-Tran et al., 2014), and a variety of adult diseases including kidney disease, prostate disease, ovarian primordial follicle loss, and polycystic ovarian disease (Manikkam et al., 2012a). As discussed below, a few animal studies have provided evidence of transmission of adverse effects to later generations.

Mechanisms that could underlie later-life effects in offspring and effects in later generations (transgenerational inheritance) could involve epigenetic processes as described at the beginning of this chapter. Research into dioxin’s potential as an epigenetic agent is in its early stages, but a few studies have suggested that dioxin has such properties that are, in significant part, linked to the AHR. Direct evidence, however, is limited to maternal exposures of the developing embryo or fetus during in utero growth, and no reports exist showing paternal TCDD exposure and later-life effects in offspring or paternally mediated transgenerational effects. Wu et al. (2004) demonstrated that TCDD exposure of mouse embryos before implantation in unexposed females resulted in epigenetic changes, including increased methylation and reduced expression of imprinted genes, which implied that early embryonic exposure alone was sufficient to alter gene expression in the resulting offspring. Transmission of effects to later generations would involve epigenetic alterations in the developing germ cells of a fetus that was directly exposed to maternal TCDD in utero, and, as noted earlier in this chapter, a recent study suggests that environmental epigenetic effects are erased during the post-fertilization DNA demethylation period (Iqbal et al., 2015). In addition, odor fear conditioning in the father could be paternally transmitted to F1 and F2 generations, and reduced DNA methylation in the responsible odor receptor gene was implicated (Dias and Ressler, 2013). Clearly, the development of more and better research models will be required to improve understanding of how transgenerational effects can be transmitted paternally (Dias and Ressler, 2014).

Results of a few recent studies support a transgenerational inheritance due to in utero exposure to TCDD. Exposing pregnant mice to TCDD (at 10 mg/kg) reduced fertility and increased premature birth in three later generations (Bruner-Tran and Osteen, 2011); effects were transmitted through both male and female offspring (Ding et al., 2011; McConaha et al., 2011). Exposing gestating female rats (F0) to dioxin (TCDD) at 100 ng/kg was recently shown to result in earlier puberty in the offspring (F1) and two later generations (F2 and F3) and to reduce ovarian follicle numbers in the females of the F3 generation; this implies transgenerational inheritance (Manikkam et al., 2012a). The F3 effects appear to be transmitted through the sperm that were initially exposed to maternal dioxin in utero. In a second paper by the same research team, additional diseases appeared later in life in the first generation (directly exposed offspring), including prostate disease in males and ovarian follicle loss and polycystic ovarian disease

in females (Manikkam et al., 2012b). Further third-generation effects were noted, including kidney disease in males and polycystic ovarian disease in females, which imply transgenerational inheritance. The latter appear to be transmitted through the sperm originally exposed to maternal dioxin in utero inasmuch as sperm DNA methylation changes were observed at 50 chromosomal sites in generations F1–F3. Testicular inflammation from TCDD exposure has also been reported to manifest in multiple generations (Bruner-Tran et al., 2014).

The zebrafish has been used as a model to examine the transgenerational effects from dioxin exposure, although different groups have reported different aspects of these effects since Update 2012. One group reported that exposing zebrafish at 3 and 7 weeks old (during sexual development) to TCDD in water for 1 hour at 50 pg/ml increased female-to-male ratios and skeletal abnormalities and reduced fertility in the F1 and F2 descendants (equivalent to F2 and F3 in mammals) (Baker et al., 2014a,b). Another group studying DNA methylation changes in the offspring of mothers fed 20 µg/kg TCDD in their food reported no changes in global methylation in offspring when looking at the total levels of DNA methylation in the genome. However, gene-specific increases or decreases in promoter DNA methylation were observed with a tiling array assay for a limited number of genes in the F1 generation. CYP1A1 transcription, a marker of TCDD exposure, was elevated in F1 offspring. Unfortunately, no F2 fish were generated from TCDD exposure because the F1 fish died 1 to 2 weeks post hatching (Olsvik et al., 2014). Further work with this model will be helpful for providing targets for mammalian biologists as they continue to probe for transgenerational effects from TCDD and the other COIs.

Another mode of epigenetic change is the modification of the spatial arrangement of chromosomes, which can influence gene expression and cell differentiation. Oikawa et al. (2008) found that TCDD, through the AHR, modifies the positions of chromosomes in the interphase nuclei of human preadipocytes.

The studies discussed above suggest that TCDD has the potential to influence the epigenome and therefore could promote changes in offspring that lead to disease later in life, although research addressing this issue in model systems is still at an early stage.

Synthesis

The epidemiologic studies designed to examine the effects of the COIs in more mature offspring have evaluated a variety of biomarkers pertaining to the neurologic, immunologic, and endocrine systems. More studies are required before conclusions can be reached as to whether such outcomes in the offspring of exposed parents are replicable. In particular, it would be of interest to obtain information on neuropsychiatric conditions, such as attention-deficit hyperactivity disorder and other clinically defined neurodevelopmental outcomes, in children who were exposed in utero. The animal literature contains evidence that

environmental agents mediated by maternal exposure affect later generations through fetal and germline modifications, but in the case of adult male exposures before conception of the next generation, there is insufficient evidence of transgenerational affects.

Conclusions

There is inadequate or insufficient evidence to determine whether there is an association between the exposure of men and women to 2,4-D, 2,4,5-T, TCDD, picloram, or cacodylic acid before conception or during pregnancy and disease in their children as they mature or in later generations. Although the results of laboratory research support the plausibility of transgenerational clinical conditions, human data are currently lacking to support an association between the COIs and such disease states in human offspring.

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