10Effects on Future Generations

Chapter Overview

Based on new evidence and a review of prior studies, the committee for Update 2012 did not find any new significant associations between the relevant exposures and adverse outcomes in future generations. Current evidence supports the findings of earlier studies that

  • No adverse outcomes in future generations had sufficient evidence of an association with the chemicals of interest.
  • There is limited or suggestive evidence of an association between the chemicals of interest and spina bifida.
  • There is inadequate or insufficient evidence to determine whether there is an association between parental exposure to the chemicals of interest and birth defects other than spina bifida, 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 Vietnam (VAO; IOM, 1994) contained a single chapter devoted to reproductive outcomes, as was the case through the publication of 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, and 2010 [IOM, 1996, 1999, 2003, 2005, 2007, 2009, 2011]). In Update 2002, the chapter's concerns were extended to include 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 the current update 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. The current chapter focuses and expands on issues related to possible adverse effects in future generations—both the children of Vietnam veterans and their offspring in turn. 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 corresponding interest in the Department of Veterans Affairs, in Update 2010 the attention of 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. It is hoped that by devoting a separate chapter to the possible “post-birth” problems of the progeny of Vietnam veterans, 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 2010 that investigated associations between parental exposure to herbicides and adverse effects on offspring, including future generations, throughout their life spans. The epidemiologic literature considered in this chapter includes studies of a broad spectrum of effects in 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, 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% to total TEQs, based on the World Health Organization (WHO) revised toxicity equivalency factors 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 potential adverse effects of herbicide exposure on men. For non-reproductive outcomes, the etiologic importance of the exposed person's sex does not play a dominant role; but for the possible transmission of adverse effects to future generations, it is critically important, from the perspective of the biologic mechanism, which parent experienced the exposure in question. 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 not only for the COIs in the VAO report series but 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 conception. With the possible 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, direct exposure of the fetus throughout gestation is possible owing to mobilization of toxicants from the mother's adipose tissue. The chapter also addresses the biologic plausibility of adverse effects on offspring mediated by male veterans through semen transmission during pregnancies that occurred after 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 FUTURE GENERATIONS

There have been few offspring studies of the four herbicides in question, particularly picloram and cacodylic acid, and those studies generally have shown toxicity only at very high doses, so the preponderance of the following discussion concerns TCDD, 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 high exposure, there may still be large amounts of dioxins stored in fat tissue, which may be mobilized, particularly at times of weight loss. That would not be expected to be the case for nonlipophilic 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, which had long been believed to consist almost exclusively of greatly condensed, transcriptionally inert deoxyribonucleic acid (DNA) for half the paternal genome (a haploid set of chromosomes). As a result, it was thought that any paternally-derived damage to the embryo or offspring would have to arise from changes in sequence or arrangement of the sperm's DNA; the fact that dioxins have not been shown to be genotoxic fostered skepticism that adverse outcomes in offspring could arise from paternal exposure to the COIs. More recently, however, it has been recognized that sperm also carry a considerable collection of ribonucleic acid (RNA) fragments (Kramer and Krawetz, 1997; Krawetz et al., 2011). Although ribosomal (rRNAs) and messenger RNAs (mRNAs) have been detected in mature sperm, as yet, any roles they may play in fertilization or development have not been delineated. Functionality has been demonstrated for several of the small RNAs found in mature sperm (Krawetz, 2005); they have been found to play critical roles in early embryonic development (Hamatani, 2012; Suh and Blelloch, 2011) and epigenetic determinations (Kawano et al., 2012). Epigenetic effects are ones that result in permanent (heritable) changes in gene expression without a change in DNA sequence arising from modification to DNA (usually involving methylation) or to other cellular components such as histones and RNAs (Jirtle and Skinner, 2007). Alterations in DNA expression arising from epigenetic modification of an individual's somatic cell lines may not be manifested for long periods of time. In epigenetic transgenerational inheritance, an alteration in the germ line 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), so this process requires exposure precisely at the time in germ-line development when epigenetic programming is being established (Skinner et al., 2010). Therefore, paternally-derived adverse outcomes in offspring associated with exposure to the COIs could be mediated not just by genetic alterations of DNA, but also by epigenetic modifications to components of sperm in addition to their DNA (Krawetz, 2005). There is also a more remote possibility, if body burden were sufficiently high, that TCDD exposure might occur through absorption of seminal plasma through the vaginal wall, which could affect gestating offspring in an otherwise unexposed mother.

A mother's (maternal) contribution to a pregnancy and offspring is obviously more extensive, 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. 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. There are two feasible pathways through which TCDD and other COIs from paternal exposures could lead to developmental and later life effects in offspring and potentially future generations. 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. Those effects would occur before conception. The other involves transmission of the contaminants to a female partner through seminal fluid during an established pregnancy, that is, after conception.

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. There is potential for TCDD to alter sperm cells of adults before fertilization through epigenetic pathways. The sperm epigenome is distinct from that of the egg (oocyte) or somatic cells (all other nongamete cells in the body). The mature sperm cell has less global methylation than somatic cells and unique DNA methylation marks (particularly on paternally imprinted genes) that put the gametes in a pluripotent state before fertilization (Hales et al., 2011). Chemical alterations of methylation foci in DNA of adult sperm have the potential to contribute to permanent effects in offspring, as demonstrated in fetal alcohol syndrome (Jenkins and Carrell, 2012a; Ouko et al., 2009). During spermatogenesis in the adult, most sperm histones are replaced by protamines, which render the sperm transcriptionally quiescent and permit extensive DNA compaction. But recent evidence has shown that some core histones are retained in human sperm at sites that are important during embryo development, so their perturbation by exogenous chemicals remains a possibility (Hammoud, et al., 2009). That is particularly important because although genome-wide DNA demethylation occurs in paternal DNA after-fertilization and would 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). Finally, despite the exclusion of almost all cytoplasm, mature sperm have been found to carry a diverse spectrum of RNAs, including mRNAs, rRNAs, and noncoding RNAs, which may affect the developing embryo (Hamatani, 2012; Krawetz, 2005; Krawetz et al., 2011; Suh and Blelloch, 2011). It has recently been demonstrated that 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). Heavy metals have been shown to 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, but dioxins have been shown to modify DNA methylation in microRNAs in somatic cells (Hou et al., 2011), so the pathway is biologically plausible.

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 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 concentrations are high, they will 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 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 (1996) study, serum TCDD was measured in 50 Vietnam veterans from Michigan who had confirmed or self-reported potential for Agent Orange exposure and had blood drawn an average of 26 years after the possible exposure. Of those, 6 had TCDD greater than 20 parts per trillion (ppt) on a lipid-adjusted basis, and this supports the idea that some veterans did have 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 measured concentrations were very low, the results documented the existence of dioxins and dibenzofurans in seminal plasma of the veterans long after possible Agent Orange exposure. Because results on serum and semen concentrations could not be linked for individual veterans and because it is unknown whether any of the subjects 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 Operation Ranch Hand specimens, however, might provide a valuable resource for comparing TCDD concentrations in serum and seminal fluid.

Furthermore, 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 that end, one must take into account several factors: the volume of seminal plasma is relatively low (1–5 mL); because of leakage, only a fraction of seminal constituents are absorbed across the vaginal wall; and dilution of absorbed chemicals in the female bloodstream (that is, in a high volume) before transmission across the placenta is estimated at 3 orders of magnitude or more (Klemmt and Scially, 2005), and this 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.

Empirical Epidemiologic Evidence on Paternal Transmission

The idea that 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. 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 offspring. 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 leukemia 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 fathers' radiation exposures before conception and an increase in leukemia among their children. However, later studies have failed to confirm that finding (Draper et al., 1997; Kinlen, 1993; Kinlen et al., 1993; Parker et al., 1993). Similarly, rigorous followup of children of atomic-bomb survivors has not demonstrated increased risks of cancer or birth defects (Izumi et al., 2003; Schull, 2003), and other studies of effects (birth defects and cancer) 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).

The committee was unable to find a single instance of epidemiologic evidence that convincingly demonstrated that paternal exposure to any particular chemical before conception resulted in cancer or birth defects in offspring. However, there are few data for addressing 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 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 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 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 paternal exposures. Because of the long half-life of TCDD and its bioaccumulation in adipose tissues, women exposed to Agent Orange in Vietnam would have 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 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, 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 6 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 disease onset later in life.

An emerging field of research referred to as the developmental basis of adult disease (Barker et al., 2012) has been investigating maternal nutritional exposures, stress, and alcohol exposure, and more recent studies have involved 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, and adult-onset cancers. Furthermore, germ cells (eggs and spermatogonia) in offspring undergo critical developmental stages during fetal life, and emerging evidence demonstrates that fetal exposures are capable of altering the germ cells epigenetically and of resulting in transmission of adverse effects to future generations (transgenerational inheritance).

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. 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

March of Dimes defines a birth defect as an abnormality of structure, function, or metabolism, whether genetically determined or resulting from an environmental influence during embryonic or fetal life (Bloom, 1981). Other terms, often used interchangeably, are congenital anomaly and congenital malformation. Major birth defects, which occur in 2–3% of live births, are abnormalities that are present at birth and are severe enough to interfere with viability or physical well-being. Birth defects are detected in another 5% of babies through the first year of life. The causes of most birth defects are unknown. Genetic factors, exposure to some medications, exposure to environmental contaminants, occupational exposures, and lifestyle factors have been implicated in the etiology of birth defects (Kalter and Warkany, 1983). 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 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 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 (Kang et al., 2000) that reported significant increases in birth defects in their offspring, did not find those results adequate to modify prior conclusions, although Congress did mandate service-related status to a number of birth defects in the children of female Vietnam veterans. 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.

TABLE 10-1. Selected Epidemiologic Studies—Birth Defects in Offspring of Subjects (Shaded Entries Are New Information for This Update).

TABLE 10-1

Selected Epidemiologic Studies—Birth Defects in Offspring of Subjects (Shaded Entries Are New Information for This Update).

TABLE 10-2. Selected Epidemiologic Studies—Neural-Tube Defects in Offspring of Subjects.

TABLE 10-2

Selected Epidemiologic Studies—Neural-Tube Defects in Offspring of Subjects.

Update of the Epidemiologic Literature

No Vietnam-veteran, occupational, or case-control studies of exposure to the COIs and birth defects have been published since Update 2010.

Environmental Studies

Since Update 2010, three studies have examined maternal exposure to the COIs in relation to congenital cryptorchidism or hypospadias; two based on a Danish–Finnish joint prospective cohort (Krysiak-Baltyn et al., 2012; Virtanen et al., 2012) and one that used the US National Birth Defects Prevention Study (NBDPS), a population-based case-control study of congenital malformations that uses a multistate surveillance systems (Rocheleau et al., 2011).

Virtanen et al. (2012) examined placental concentrations of dioxins and PCBs in relation to congenital cryptorchidism in a nested case-control study within the joint prospective Danish–Finnish cohort study of the incidence of and risk factors for congenital cryptorchidism and hypospadias. Boys born in 1997–2001 in Copenhagen were examined for cryptorchidism at birth and at the age of 3 months. In preterm boys who had undescended testis, cryptorchidism was diagnosed only if the testis remained undescended at the expected date of delivery. Midwives collected and froze placentas immediately after birth. The study included 56 Finnish cases and 56 controls that were individually matched on date of birth (± 2 weeks), parity, gestational age (± 1 week), smoking during pregnancy, and maternal diabetes. It also included 39 Danish subjects and 129 controls that were not matched on the above factors. Concentrations of 17 PCDD or PCDFs and 37 PCBs were measured and presented as total TEQs and as individual congeners. There were no significant differences between cases and controls in either the Finnish or Danish samples with respect to dioxin TEQs. Although there were some isolated and country-specific significant associations, none was replicated in all the countries, and concentrations of some congeners in controls exceeded those in cases. In a similarly designed study, Krysiak-Baltyn et al. (2012) examined breast-milk concentrations of PCBs and dioxins in relation to congenital cryptorchidism. Of the COIs measured, only dioxin-like OctoCDF concentrations in cases exceeded those in controls, and only in the Danish subset.

Using a job–exposure matrix to estimate maternal herbicide exposure from conception through the first trimester of pregnancy, Rocheleau et al. (2011) examined the association of herbicide exposure with hypospadias in the NBDPS. Affected children and fetuses were identified through active case ascertainment by each surveillance program. Controls (1,496) were a random sample of all unaffected live births in the areas covered by the state-based surveillance systems, and cases (647) had a diagnosis of second- or third-degree hypospadias. Maternal interviews were conducted no later than 24 months after delivery. The following covariates were examined for evidence of confounding: maternal age; parity; history of miscarriage, singleton, or multiple pregnancy; gestational age and birth weight of the index infant; maternal alcohol consumption or smoking during or before the first trimester; use of a folic acid–containing supplement; prepregnancy body mass index; and several sociodemographic characteristics. The overall participation rate was about 70%. In general, participants exposed to herbicides were also exposed to insecticides or fungicides. In a model adjusted for all other pesticide classes, periconception herbicide exposure was not associated with second- or third-degree hypospadias (odds ratio [OR] = 0.99, 95% confidence interval [CI] 0.47–2.10), but there were very few exposed cases (36).

Ren et al. (2011) conducted a case-control study of neural-tube defects (NTDs) in subjects recruited from four rural counties of the Shanxi Province in China in 2005–2007. Cases were identified through a population-based birth-defects surveillance program. Healthy controls were individually matched to cases on sex, hospital of birth, mother's county of residence, and date of the mother's last menstrual period ("as close as possible"). Maternal interviews were conducted within 1 week of pregnancy termination or delivery to ascertain information on periconception use of folic-acid supplements; smoking exposure; exposure to pesticides, solvents, and heavy metals; other environmental exposures; and a variety of demographic, lifestyle, and reproductive history information. Placentas were collected at delivery or termination of NTD-affected pregnancies and measured for concentrations of polycyclic aromatic hydrocarbons, organochlorine pesticides, PCBs, and lipids. Models were adjusted for matching factors, in addition to maternal occupation, age, educational level, parity, folic-acid supplementation, passive smoking, and fever or influenza during pregnancy. Of the eight PCB congeners measured, six exhibited some dioxin-like activity (mono-ortho PCBs 105, 118, 156, 157, 167, and 189), but the measures of association were provided only as a sum, including also 206 and 209. Overall, there were no differences in the median placental concentrations of the total PCB sum between NTD cases (0.90 ng/g of lipid) and controls (0.87 ng/g of lipid).

Biologic Plausibility

2,4-D has been previously shown to be a teratogen, although at exposures that exceed maternal renal clearance, which are not relevant to Agent Orange exposure. A new 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. It is well established that TCDD is a potent teratogen in all laboratory species that have been studied, although the pattern of birth defects that are produced is often species-specific. Since Update 2010, studies have investigated the mechanisms underlying various TCDD-induced birth defects in rodents and other animal models, 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; Yoshioka et al., 2012; Yuan et al., 2012). Those mechanisms have not been fully elucidated, but it has been demonstrated that TCDD-induced birth defects require the aryl hydrocarbon receptor (AHR) but do not require induction of cytochrome P4501A1 (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, activation of the AHR by TCDD during development appears to be a key first step in mediating TCDD's developmental toxicity. 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. It has been shown that antioxidant treatment provides protection against some TCDD-induced teratogenicity; this suggests that reactive oxygen species might be involved in the pathways that lead to these structural changes (Jang et al., 2008). A few new studies indicate that stem cells and organ-specific progenitor cells may be direct targets and that maternal TCDD exposures interfer with proliferation and cell differentiation through the AHR and result in defects in organ morphogenesis (Latchney, 2011; Neri, 2011). Few laboratory studies of potential male-mediated developmental toxicity (and, specifically, birth defects) attributable to exposure to TCDD and herbicides have been conducted. Feeding of simulated Agent Orange mixtures to male mice produced no adverse effects in offspring (Lamb et al., 1981).

Synthesis

Embryonic and fetal development in rodents is sensitive to toxic effects of exposure to TCDD and dioxin-like chemicals. It is clear that the fetal rodent is more sensitive to 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 used extremely low exposures. The four studies since Update 2010 that have assessed exposure to relevant chemicals and congenital malformations all examined only maternal exposure, which is of little relevance to the majority of Agent Orange–exposed veterans. Furthermore, those environmental 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.

Conclusions

There was one new study of the relationship between maternal exposure to dioxin-like, mono-ortho PCBs and NTDs in offspring, which found no association, and there were no new studies of parental exposure to 2,4-D, 2,4,5-T, TCDD, cacodylic acid, or picloram and spina bifida in offspring. The committee concludes that the evidence of an association between exposure to the COIs and spina bifida is still limited or suggestive. The evidence of an association between exposure to the COIs and other birth defects is inadequate or insufficient.

CANCERS IN OFFSPRING

The American Cancer Society (ACS) estimated that 11,630 children less than 15 years old will receive a diagnosis of cancer in the United States in 2013 (ACS, 2013). 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% in the 1970s to more than 80% in 2013. Despite those advances, cancer remains the leading cause of death from disease in children less than 15 years old, and 1,310 deaths were projected for 2013 (ACS, 2013).

Leukemia is the most common cancer in children, accounting for about one-third of all childhood cancer cases. In 2010, ACS anticipated that almost 3,317 children would receive a leukemia diagnosis (ACS, 2010). Of those, almost 2,000 would have acute lymphocytic leukemia (ALL); most of the rest would have acute myeloid leukemia (AML). AML (International Classification of Diseases, Ninth 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 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 leukemia as part of the discussion of adult cancer.

The second-most common group of cancers in children are those 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. 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 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, and Update 2010 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.

TABLE 10-3. Selected Epidemiologic Studies—Childhood Cancer.

TABLE 10-3

Selected Epidemiologic Studies—Childhood Cancer.

Update of Epidemiologic Literature

No Vietnam-veteran, occupational, or environmental studies of exposure to the COIs and childhood cancer have been published since Update 2010.

Case-Control Studies

Two childhood-leukemia studies examined the relationship of herbicide exposures and leukemia risk. Chokkalingam et al. (2012) conducted a population-based epidemiologic study of 377 ALL cases and 448 controls in the Northern California Childhood Leukemia Study and examined association with self-reported exposure to indoor and outdoor household pesticides overall and in subgroups that had hypothesized susceptibility genotypes in 42 xenobiotic transport and metabolism genes. With no adjustment for confounders indicated, they found a borderline-significantly-increased risk of ALL with outdoor herbicide use before birth (OR = 1.46, 95% CI 1.04–2.04) and with indoor insecticide use (OR = 1.29, 95% CI 0.97–1.72).

Slater et al. (2011) examined infant leukemia risk in the Children's Oncology Group study associated with maternal exposure to herbicides at any time during the period from 1 month before conception throughout pregnancy, during only the preconception period, and during only the pregnancy. They found no significant associations.

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 inheriting a genetic predisposition, which by itself could increase the development of cancer or the likelihood of developing cancer after future exposure to a carcinogen; the mother or father would transmit either an acquired genetic defect or an epigenetic alteration that predisposed the child to cancer. Alternatively, a maternally-mediated increase in susceptibility to childhood cancer could result from direct exposure of a child in utero or via lactation to a xenobiotic that induces epigenetic alterations that increase cancer susceptibility or is itself carcinogenic.

It has been shown that 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). 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). Developmental epigenetic alterations may be involved in the prenatal effects. TCDD has been shown to suppress the expression of two tumor-suppressor genes, p16Ink4a 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).

Although there is no direct evidence from animal models that TCDD increases the risk of childhood cancers, such as acute leukemia and germ-cell tumors, 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; this suggests that childhood leukemias begin before birth, perhaps due to maternal exposures to genotoxic xenobiotics.

Synthesis

Two case-control studies considered childhood leukemias and herbicide use. One found a marginally significantly increased risk of ALL in association with maternal herbicide use before conception, and the other saw no increases in childhood leukemias related to maternal herbicide exposure shortly before or during pregnancy. No new epidemiologic evidence specifically concerning the COIs and childhood cancers has been published since Update 2010.

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 is 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 cancer (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. 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.

Conclusions from VAO and Previous Updates

The potential effect of maternal and paternal exposure of Vietnam veterans to herbicides on the development of disease other than cancer in their 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 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 the studies discussed in this section of Update 2010 would be applicable only to children born to female Vietnam veterans during or after their deployment in Vietnam.

Changes Detected in Children After Parental Exposure

Thyroid Hormone Concentrations

Since Update 2010, there has been one additional epidemiologic study of childhood thyroid hormone concentrations associated with perinatal exposure to dioxins and dioxin-like PCBs. Leijs et al. (2012) conducted a followup study of 33 children 14–19 years old in whose mothers' breast milk PCDD or PCDF exposure was determined. All children were born in the Amsterdam–Zaandam region. Spearman's correlations were calculated by comparing PCDD, PCDF, and dioxin-like PCB TEQs with childhood triiodothyronine (T3), thyroxine (T4), free thyroxine (FT4), thyroxine-binding globulin (TBG), and thyroid-stimulating hormone (TSH) concentrations. Laboratory methods for measuring thyroid hormone were not provided. There was no correlation between perinatal dioxin exposure and T3, T4, FT4, TBG, or TSH. There was a significant correlation between dioxin-like PCB TEQs and childhood T3, but the magnitude was not provided; the significance level was p = 0.047. Those results conflict with the results of the Nagayama et al. (1998) study; however, the Leijs et al. (2012) study was small, and no covariate adjustment was performed for potentially-important factors, such as the child's age and sex, the mother's age, and the mother's smoking or alcohol consumption during pregnancy.

Cognitive or Motor Development

Since Update 2010, there has been one additional study of infant neurobehavioral development in relation to prenatal dioxin exposure. Nishijo et al. (2012) examined the association between dioxin exposure and infant growth and development in 210 mother–infant pairs that resided in dioxin-contaminated districts near the Da Nang airbase in Vietnam. Full-term babies from uncomplicated deliveries were recruited in 2008–2009. Breast milk was collected 1 month after birth and analyzed for 7 PCDDs and 10 PCDFs. Maternal interviews provided detailed covariate data, and pregnancy and delivery information was obtained from the obstetricians. All infants were breastfed until 4 months after birth. The duration of residence in the contaminated districts was directly related to PCDD and PCDF TEQ exposure quartile and maternal age. In boys, statistically-significant decrements in expressive communication skills as measured by the Bayley Scales of Infant and Toddler Development III (BSID-III) at the age of 4 months were noted in the 4th quartile of exposure relative to the 1st. Although the differences were not statistically significant, infants of both sexes in the highest quartile of prenatal exposure exhibited lower cognitive scores on the BSID-III (about 6 points than the 1st quartile in boys and 4 points in girls) and lower total motor scores (about 4 points in boys and 3 points in girls). However, measures of neurodevelopment in very early life are generally unstable.

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

Since Update 2010, there has been one additional study of allergies and infections during infancy in relation to prenatal exposure to dioxin-like compounds. Miyashita et al. (2011) examined allergies and infections in 364 mother-infant pairs enrolled during 2002–2005 in the Hokkaido Study on Environment and Children's Health (Sapporo, Japan). Third-trimester maternal blood concentrations of PCDDs, PCDFs, and dioxin-like PCBs were measured, and total maternal dioxin TEQs were calculated. Covariates (including exposure to environmental tobacco smoke, maternal education, annual household income, and maternal dietary intake of fish and meat during pregnancy) were assessed through maternal interviews. Maternal interviews also provided information about hospitalization or medical treatment of infants for asthma, eczema, other allergic diseases, otitis media, febrile seizures, respiratory syncytial virus infection, and other diseases from birth to the age of 18 months. A modified version of the International Study of Asthma and Allergies in Childhood (ISAAC) questionnaire was administered. Development of allergies or infections in infants was defined as having had a doctor's diagnosis, hospitalization, or medical treatment between birth and the age of 18 months. Asthma was expanded to include cases in which the mother gave positive responses to all questions on the modified ISAAC questionnaire. There were no associations between maternal exposure and childhood food allergy, eczema, or asthma, although there was a weak positive trend of increased risk of asthma with increasing exposure to PCDFs (p = 0.059). There was also a weak positive association of third trimester PCDF concentrations and increased risk of otitis media (overall trend p = 0.027); compared with the children of women in the lowest quartile of exposure, the children of those in the highest quartile had 2.5 times the risk of having a confirmed case of otitis media (95% CI 1.07–5.88) in multivariate adjusted models. The effect was more pronounced in male infants, who showed an indication of a dose–response relationship, with an increase in risk in the highest quartile for dioxin activity from furans (OR = 3.80, 95% CI 1.09–13.18) and for total dioxin activity (OR = 4.44, 95% CI 1.20–16.45). There were also significant congener-specific associations with otitis media for octachlorodienzo-p-dioxin (OCDD) (all quartiles relative to the 1st quartile), 2,3,4,7,8-pentachlorodibenzofuran (4th quartile relative to 1st quartile), dioxin-like non-ortho PCB 77 (4th quartile relative to 1st quartile), and dioxin-like mono-ortho PCB 157 (2nd quartile and 4th quartile relative to 1st quartile). Those findings provide some support for immunotoxic effects of dioxin-like compounds when the mother is exposed.

Jusko et al. (2011) reported on PCB exposures in Eastern Slovakia, examining concentrations in maternal and cord serum and immunoglobulin concentrations in offspring. No association between immunoglobulin concentrations and exposure was noted.

Offspring Reproductive Function

Since Update 2010, there have been three epidemiologic studies of maternal or perinatal exposure to dioxins and dioxin-like PCBs in relation to child reproductive development. Humblet et al. (2011) examined the association with maternal dioxin exposure in the highly-contaminated region of Chapaevsk, Russia, which until 2003 was the site of chlorinated-chemical production at the Middle Volga Chemical Plant. A mother's serum concentration 8–9 years after pregnancy served as a surrogate measure of her son's in utero and lactational exposure. The study investigated 444 mother–son pairs (89% of the 499 peripubertal boys in the entire cohort) that were recruited when the boys were 8–9 years old by using the townwide health-insurance information system in 2003–2005. At study entry, a physical examination was conducted on each boy, maternal and child blood samples were drawn for measurement of TEQs and total PCB concentrations, and an extensive interview was administered. One of the study investigators (blinded to the subjects' dioxin concentrations) conducted all pubertal staging at study entry and annually thereafter. A carefully-considered covariate adjustment plan was implemented in this high-quality study. No association of maternal serum total TEQs was seen in any of the three indexes of pubertal onset measured. In a subset of boys who breastfed for at least 6 months, a dose-related delay (relative to those who breastfed less) in pubertal onset was seen with increasing quartiles of maternal TEQs. Overall, the study does not provide particularly strong or consistent evidence of an association between perinatal TEQ exposure and pubertal onset among boys.

Reproductive function in sons was also investigated by Mocarelli et al. (2011); 78 men 18–26 years old who were born to women living in the most dioxin-polluted areas near Seveso, Italy, were eligible for the study. After refusals and exclusion for varicoceles, only 39 subjects (50%) remained, but previously-gathered information on all the mothers demonstrated that nonparticipation was not associated with the mothers' serum concentrations or with whether the sons were breastfed. Of the 39, 21 were breastfed (and so received both in utero and postnatal exposure via their mothers), and 18 were formula-fed (and so received only in utero exposure from their mothers). At-home semen samples were collected from the sons and graded according to the WHO (WHO, 1999) recommendations. At the same visit, fasting blood samples were obtained, and they were measured for follicle-stimulating hormone (FSH), inhibin B, serum 17-β-estradiol, luteinizing hormone, and testosterone. Maternal serum TCDD measurements were taken from maternal serum frozen since 1976–1977. Of consecutive blood donors, 123 men matched for age and socioeconomic status whose mothers had not resided in the contaminated area were asked to be controls, and 58 (47%) participated. Hormone data were adjusted for body mass index (BMI), smoking behavior, age at the time of test, chemical exposure, and alcohol use. Sperm-function models were also adjusted for educational level, employment status, and abstinence time. Seveso-exposed men had a lower adjusted mean sperm concentration than the comparison population (46.2 × 106/mL vs 81.0 × 106/mL, p = 0.01), lower total sperm count (139.2 × 106 vs 229.9 × 106; p = 0.03), and lower progressive motile sperm count (50.6 × 106 vs 90.5 × 106; p = 0.05). These and other associations (sperm progressive motility, FSH, and inhibin B) appeared to be substantially modified by whether the man had been breastfed in childhood; the effects were strongest in, and to some degree limited to, men who had been breastfed. This indicates that early-life exposure via breast milk had more of an impact on these fertility-related outcomes in the later lives of the sons than did in utero exposure, but breastfeeding is a relevant mode of maternal exposure for the children of female Vietnam veterans. Those results suggest an effect of early-life exposure on adult reproductive function. Their reliability depends on the assumption that the Seveso-exposed and comparison populations are similar on all factors other than exposure, but the data provided indicate moderate differences in the sons' ages and duration of breastfeeding, while the covariate data provided are too limited for a full evaluation of the similarity of the groups. The results might have been enhanced by analyses using the mothers' measured dioxin concentrations.

Finally, Su et al. (2012) followed up 56 children (23 boys and 33 girls) from the Taiwanese mother–child birth cohort previously described (Chao et al., 2004; Wang et al., 2004, 2005). Children were stratified into low and high median placental PCDD, PCDF, and PCB exposure groups according to their mother's overall median exposure. The children's 8-hour fasting blood samples were obtained at followup and analyzed for testosterone, estradiol, luteinizing hormone, FSH, triglyceride, cholesterol, and insulin. There were few associations overall between dioxins and dioxin-like PCBs and hormone concentrations, other than higher median estradiol concentrations (3.0 ng/dL vs 1.8 ng/dL) in children whose mothers had lower total TEQs than in children whose mothers had higher total TEQs (p = 0.003). However, the comparisons were not adjusted for plausible confounders.

Biologic Plausibility

As reported in Update 2010, 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., 2011; Prescott, 2011; Puga, 2011; Takeda et al., 2012). Results of several new studies also support the idea. Using two mouse models, investigators showed that prenatal TCDD (2.5–5.0 mg/kg of body weight) 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 μg/kg of body weight) reduced brain developmental myelination and compromised remyelination potential in adults (Fernández et al., 2010), and in utero TCDD in mice alters neural progenitor differentiation (Mitsuhashi et al., 2010). However, a recent study suggested that, unlike murine neurospheres (which represent neural progenitor cells), human neurospheres were nonresponsive to TCDD because of lack of the AHR receptor—an indication of species specificity in response (Gassmann et al., 2010). Perinatal TCDD (0.2–0.4 μg/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, and this supports the idea of continued later-life thyroid hormone disturbances (Ahmed, 2011). 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. Direct evidence, however, is limited to maternal exposures of the developing embryo or fetus during in utero growth, and there have been no reports on paternal TCDD exposure and later-life effects in offspring or paternally-mediated transgenerational effects. As reported in Update 2010, 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. The germ-line epigenome modified either through altered DNA methylation or through core histone modifications would be permanent (that is, would escape the normal erasure of an imprinted gene) and would be transmitted over several generations.

Results of a few recent studies support a transgenerational inheritance due to in utero exposure to TCDD. Exposure of pregnant mice to TCDD (at 10 μg/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). Exposure of 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 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 (Manikkan et al., 2012b). Further third-generation effects were noted, including kidney disease in males and polycystic ovarian disease in females, and 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.

Another mode of epigenetic change is modification of the spatial arrangement of chromosomes, which can influence gene expression and cell differentiation. Oikawa et al. (2008) have 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.

Synthesis

The epidemiologic studies designed to examine effects of the COIs in more-mature offspring have evaluated a variety of biomarkers pertaining to the neurologic, immunologic, and endocrine systems. Most have not examined defined clinical conditions, although data on associations with otitis media (Miyashita et al., 2011; Weisglas-Kuperus et al., 2000) and impaired fertility in adult sons of exposed females (Mocarelli et al., 2011) are emerging. More studies that examine those and other end points are required. In particular, it would be of interest to obtain information on neuropsychiatric conditions in children who were exposed in utero, such as attention-deficit hyperactivity disorder and other clinically-defined neurodevelopmental outcomes. The animal literature contains evidence that environmental agents mediated by maternal exposure affect later generations through fetal and germ-line 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 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 results of laboratory research support the plausibility of transgenerational clinical conditions, the body of human data is insufficient to support an association between the COIs and such disease states in human offspring.

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Footnotes

1

Throughout this report, the same alphabetic indicator after year of publication is used consistently for a given reference when there are multiple citations by the same first author in a given year. The convention of assigning the alphabetic indicators in order of citation in a given chapter is not followed.