The study of generational health effects is a complex, evolving, and fast-paced area of research. Understanding the heritability of the health effects that may result from environmental exposures, particularly those that may occur in a war zone, requires a holistic approach involving multiple avenues of research to address data gaps and expand our knowledge of the mechanisms of inheritance that may affect future generations. The Volume 11 committee believes that to assess the potential long-term generational health effects of deployment exposures, a range of basic research using animal and cellular or mechanistic models will be necessary to complement the health monitoring plan and epidemiologic studies discussed in Chapter 9, thus encompassing a state of the science transgenerational study. This basic and translational research can help elucidate the genetic and epigenetic mechanisms that may be responsible for generational health effects in humans.
The Volume 11 committee recognizes that many of the research efforts described in this chapter cannot be undertaken by a single organization. The required competencies will be spread across numerous organizations, from governmental organizations to academic institutions and private entities. Whenever appropriate, the opportunities for collaboration and coordination across organizations should be explored and embraced. Two collaborations are exploring children’s health or the epigenetic markers of exposure. One is the National Institute of Environmental Health Sciences’ (NIEHS’s) Children’s Health Exposure Analysis Resource (CHEAR), an “extramural research community access to laboratory and data analyses that add or expand the inclusion of environmental exposures in children’s health research” (NIEHS, 2018). The second is the Toxicant Exposures and Responses by Genomic and Epigenomic Regulators of Transcription (TaRGET) II Program, sponsored by NIEHS, that seeks to explore changes in “epigenomic marks across target tissues/cells (those adversely affected by environmental exposures) and surrogate tissues/cells (those that are easily accessible and reflect the environmental exposures) using mouse models of environmentally relevant exposures” (Wang et al., 2018). Other consortia that are working to understand the human epigenome include the National Institutes of Health Roadmap Epigenomics Mapping Consortium that aims to develop a “public resource of human epigenomic data to catalyze basic biology and disease-oriented research” and that has mapped the epigenomes of more than 100 types of cells and tissues (NIH, 2018), and the International Human Epigenome Consortium that has a goal of
understanding “the extent to which the epigenome has shaped human populations over generations and in response to the environment” (IHEC, 2018). The Department of Veterans Affairs, the Department of Defense, the National Institutes of Health, and other government agencies are part of the Armed Forces Institute of Regenerative Medicine (AFIRM) network which is seeking to develop advanced treatment options for severely wounded service members using an interdisciplinary, multi-institution approach (MRMC, 2018). These and other such consortia and networks provide examples of mechanisms for taking advantage of new and existing research resources.
This chapter outlines some of the data and knowledge gaps of the molecular, biochemical, cell, and tissue basis for generational effects. It also identifies mechanistic (including in vitro models using human tissues) studies in animal models of human health outcomes that might help to fill data gaps and improve our understanding of how deployment exposures affect the health of veterans and their descendants.
During its review of the epidemiologic and animal toxicology literature described in Chapters 4 through 7, the Volume 11 committee identified research on generational health effects from exposures that are potentially relevant to Gulf War and Post-9/11 veterans, such as organophosphate pesticides. The committee also observed many data gaps in the research on the effects of the veterans’ exposures on their children, with even less known about effects on grandchildren. As was shown in Chapter 8, determining whether a veteran’s exposure to a toxicant causes health effects in his or her descendants is a complex problem. Researchers are beginning to search for human generational health effects and to devise the lengthy and costly epidemiologic studies needed to properly detect this phenomenon. Although targeted studies are being designed, it may be years or even decades before the effects (if any) of parental exposures become evident in a subsequent generation. Furthermore, health monitoring programs and epidemiologic studies may not capture rare events that occur in only a few descendants, that result from a narrow range of exposures (e.g., at very high or low doses), or that occur only in those instances where specific genetic profiles interact with specific exposures. To address these and the many other complexities inherent to human studies, medical researchers can turn to basic research to provide critical information on the heritability of the health effects of parental exposures across generations and the genetic and epigenetic mechanisms that may be involved.
Studies in model systems—in vitro systems that use cells and tissues as well as whole animals—where both genetics and exposures can be controlled, and generational animal studies that can be conducted in days or months rather than in the years and decades required for human studies, are powerful research approaches that can complement and inform human studies. Such studies may detect subtle effects and can follow genetic or epigenetic changes at the molecular, cellular, and whole-animal levels, including effects on germ cells.
No individual study in a model system is adequate to identify or define human risk; such studies must be complemented by an understanding of the similarities and differences between species in their biology and their responses to a toxicant. Furthermore, the results of health effects studies in model organisms must be extrapolated to humans if they are to be used to enhance our understanding of the reproductive, developmental, or generational effects of toxicant exposures across the exposure → effect → outcome continuum. Understanding the range of biological diversity across species can help with this extrapolation. For example, it has been shown that the gene for the aryl hydrocarbon receptor that mediates dioxin toxicity exhibits differences in sensitivity between mouse strains, between rats and mice, and between the model species and humans. Understanding these differences makes it possible to estimate human risk from rodent studies. For example, in its assessment of dioxins, the U.S. Environmental
Protection Agency (EPA) stated that “animal data show a wide range of species sensitivity to dioxin for a given developmental or reproductive endpoint” (EPA, 2012). For some endpoints, data show that human sensitivity is comparable to that of experimental animals (e.g., semen morphology), but for other endpoints, humans are insensitive compared to other species (e.g., cleft palate) (EPA, 2012). Although mice and rats are the most commonly used species, there can be differences in the response among different species and strain of these model animals. For example, Fischer 344 and Wistar rats exposed to trichloroethylene during gestation had an increased risk of decreased fetal weight, live birth weights, and postnatal growth, but Sprague Dawley rats did not (EPA, 2011). More expedient organism models, such as fruit flies, round worms, and zebra fish, are more easily used to examine system- or organism-wide effects and potential target tissues of effect, but they may not fully mirror the effects in humans.
To help distinguish between the effects of specific deployment exposures and the effects of other lifecourse exposures on veterans and their descendants, it is necessary to develop a better understanding of how the exposures and their timing (e.g., the age of exposed individuals or pregnancy status) affect an individual’s genome and the epigenome (see Figure 9-1). Other questions include how such genetic and epigenetic alterations are inherited through the germ line and how exposure-related adverse health outcomes are defined and identified in subsequent generations. It is in these areas where basic research may be most valuable, as it provides a unique opportunity to uncover the targets and mechanisms of action and to link exposures to adverse outcomes in a causal way. Model organisms are well suited for conducting studies of toxicants across multiple generations because investigators are able to carefully control for other variables, such as diet, that can also influence effects.
Thus, the Volume 11 committee gives high priority to a basic research program that examines all aspects of the exposure → effect → outcome continuum and, where possible, that does so in an integrative manner. This research might begin with identifying targets and mechanisms in order to identify biomarkers of exposure and effect for a toxicant; exploring factors that underlie individual variation in response to exposures, such as the impact of sex differences on response to a toxicant; and identifying which cell types (e.g., somatic cells, eggs, sperm) are most susceptible to specific exposures. This research should also include developing the following: fit-for-purpose models and reagents for generational studies; genetically engineered induced pluripotent stem cells (iPSCs); validated models (both animal- and cell-based) that are most appropriate for studying specific exposures; and methods and databases for acquiring, curating, and analyzing the resulting datasets. Each of these areas for basic research on lifecourse exposures is described below.
Biomarkers The identification, development, and validation of biomarkers of exposure, effect, and susceptibility, including “omics” biomarkers (RNA, DNA, proteins, metabolites, and lipids). Foundational work for such biomarker studies will also be needed and may include studies to explore how a particular agent may affect the genome/epigenome and manifest as adverse health effects in descendants. Studies using human materials (e.g., urine), animal models (mammalian and nonmammalian animals such as mice and Caenorhabditis elegans, respectively), and appropriate cell-based (e.g., iPSCs) systems would all reasonably be expected to contribute to the development of biomarkers of exposure and to our mechanistic understanding of multigenerational inheritance and of adverse reproductive and developmental health effects.
The derivation of an individual cell type from a person’s own iPSCs would enable a direct personalized assessment of changes to that person’s genome. At the time of a person’s enlistment
- into the military or prior to deployment, iPSCs could be collected from tissue biopsies (e.g., skin, bone marrow, or adipose tissue), and stored for later analysis (Ohnishi et al. 2012). When required, cells could be differentiated—for example, into the particular cell type that is affected in the individual being examined—and then tested directly. The use of iPSC makes it possible to model an individual’s response to a toxicant, with the advantage of directly testing the effect of exposure on a cell type and having a specific genotypic response for that individual. Reducing this process to routine practice and automating it would require substantial development, but it would provide personnel with baseline genomic information upon which an effect could be tested or confirmed.
- Target cells Understanding which cells and organs are vulnerable to specific exposures, how cell type influences genetic/epigenetic changes induced by specific exposures (i.e., which cells and tissues are at risk), and which tissues and organs exhibit an alteration linked to adverse health outcomes in descendants. Studies in this area might focus on the timing of the epigenetic programming of gametes to determine windows of susceptibility for generational effects; fundamental molecular and cell-based studies to elucidate the mechanisms underlying epigenetic reprogramming or other pathways for generational inheritance; studies of the combined effects of stress or heat exposure with specific toxicants or mixtures of toxicants or deployment-related mixtures; and studies using surrogate tissues such as blood, saliva, urine, or sperm to inform the exposures, mechanisms, and outcomes in target tissues and organs. Research efforts in this area should coordinate with and be informed by ongoing studies being conducted by the TaRGET consortium and the other consortia working on mapping the human epigenome.
- Data analysis and experimental design Development of best practices for designing robust generational studies across the exposure → effect → outcome continuum, including analysis of complex data sets generated by high-content omics technologies (e.g., the ENCODE consortium1) and those derived from observational studies in humans, which must include consideration of their many potential confounding variables. Within the context of assessing lifecourse exposures, confounders may include the following: single exposures to mixtures of toxicants, other exposures experienced by individuals over their lifecourse (e.g., occupational, recreational), potential lack of specificity in biomarkers used to measure specific exposures, and a potential inability to link the biomarker to an adverse health outcome in either the exposed parent or the child. Accounting for these confounders can best be accomplished with robust analytical tools. With the help of such tools, the joint influences of these variables can be identified and parsed so as to clarify the influence of the variable of interest. These new methods include neuronal nets, machine learning, and artificial intelligence to develop and validate statistical methodology and software—as well as platforms, pipelines, and databases—to analyze and substantiate the data. The computational studies that follow will inform and ensure reproducibility of studies designed to address these knowledge gaps.
- Sex-specificity The identification of—and discovery of the mechanisms responsible for—sex-specific differences in sensitivity to specific exposures, in effects on the genome and epigenome, and in adverse health outcomes on reproduction and in descendants. Studies that would be considered responsive to these research needs could include, for example, a comparison of the
1 ENCODE (the Encyclopedia of DNA Elements) is a program supported by the National Human Genome Research Institute to identify and characterize all functional elements of the human genome and to support the development of new technologies for high-throughput data (https://www.genome.gov/10005107/the-encode-project-encyclopedia-of-dna-elements; accessed June 13, 2018).
- effects of an exposure on male versus female germ lines and the use of sex-specific iPSCs to uncover germ-cell determinants of susceptibility and generational inheritance.
Chapters 4 through 7 described the evidence for the reproductive and developmental effects of deployment-related exposures that could occur in veterans or their offspring. Because there were so few studies in veterans themselves and virtually no new studies of their children, the Volume 11 committee considered other studies of humans exposed to the agents of concern, such as occupational and residential cohorts. Summary data presented in Tables 8-1 and 8-2 show that for the majority of agents of concern, there is inadequate/insufficient evidence linking exposures to reproductive or developmental effects or establishing the absence of such a linkage. Therefore, more data are needed to make casual inferences linking exposures to adverse outcomes. Responsive studies should focus on deployment-specific exposures (single-agent or mixtures), although reproductive or multigenerational studies using surrogate exposures may be adequate if they sufficiently address the research needs identified in this report. The knowledge gaps related to the adverse reproductive effects in veterans and developmental effects in offspring of these exposures include the following:
- Windows of susceptibility It is understood that exposures across the lifecourse have the potential to cause adverse health effects in exposed individuals. What is less clear is whether exposures at certain times during an individual’s life are more able to affect subsequent generations than exposures at other times. Thus, research is needed to identify the times at which exposures have the greatest potential to affect subsequent generations. A related issue is whether the effects of exposures differ between men and women. Because germ-cell maturation differs between the male and female germ line, it is possible that exposures during deployment will affect generational inheritance differently for men and women. Male veterans whose partners conceive will potentially have preconception exposures, whereas female veterans may have preconception exposures or exposures during early pregnancy (i.e., the first trimester, as it is unlikely that female veterans would have deployment exposures later in pregnancy) or both. Thus, it is important to delineate those times during the lifecourse when there is susceptibility to specific exposures and whether those times correspond to windows of susceptibility for modifying the genome/epigenome in a way that will affect the next generation and possibly the generations after that. Unlike human epidemiologic studies, basic research in animal models provides an accessible model for precisely identifying the windows of susceptibility. However, caution must be exercised when designing the experiments and interpreting the results because the reproductive and developmental cycles in different species do not have a one-to-one correspondence with human reproductive and developmental cycles. Thus, it is critical to select the animal model and the timing of the exposure to reflect the reproductive or developmental effects of concern. Research into which effects or mechanisms define these windows of susceptibility will also be important.
- Dose–response Although there are few measurements of deployment exposures from Vietnam, the Gulf War, or even the Post-9/11 conflicts, dose–response studies can help identify effects that may result from a range of exposures. The studies can also help elucidate information on how the agent is metabolized in the body. For agents that bioaccumulate, such as dioxins that accumulate in adipose tissue, determining exposure may not be a straightforward measurement.
- The half-life of an agent in the body is also important. Agents that are metabolized quickly, such as organophosphate pesticides, may be difficult to detect in the body or in urine after the exposure ceases, making biomarkers of exposure elusive. Such difficulties are compounded when studying the internal dose to a fetus or germ cells.
- Acute versus chronic exposures The length, location, duties, and number of deployments that a veteran experienced will vary depending on the veteran’s service branch, military occupation specialty, particular war zone (e.g., Vietnam, Gulf War, Post-9/11), and mission. Some exposures may be acute (e.g., a 1-day exposure to sarin from the demolition of the Khamisiyah munitions depot), others may be chronic (e.g., occupational exposures, such as fueling vehicles or cleaning equipment with solvent), and others may be intermittent (such as exposures to the black smoke from the burn pit at Joint Base Balad). At this time, the environmental measurements of the agents of concern during each deployment are minimal. Animal studies will be particularly helpful in determining the possible long-term reproductive and generational effects from acute versus chronic and intermittent exposures, as data for human exposures are, for the most part, not available. These limitations were evident in much of the epidemiology and basic research cited in Chapters 4 through 7.
- Mixtures How exposures to mixtures—mixtures of chemicals as well as co-exposures to chemicals and other environmental factors, including stress—affect reproductive and generational outcomes is another important knowledge gap. As described in Chapter 2 under Committee’s Approach, identifying mixtures and their variations is a significant data gap for distinguishing the effects of deployment exposure from lifecourse exposures in both veterans and their descendants. Many deployment exposures are mixtures, such as the smoke from burn pits, and these exposures may be compounded by frequent exposures to other mixtures such as pesticides in and around living quarters, or to individual toxicants such as pyridostigmine bromide. Although they are not specific to reproductive or developmental effects, animal studies have attempted to replicate deployment exposures to mixtures, such as to a combination of permethrin, DEET, and pyridostigmine bromide, in an effort to produce the symptoms of Gulf War illness. Numerous organizations have active programs that seek to understand how best to assess the toxic effects of mixtures. However, the bulk of the research on mixtures has focused on effects in the exposed individual with little information on inter- or transgenerational effects. While progress is being made in this area, much work remains to be done.
Both genetic and epigenetic alterations induced by environmental exposures can cause changes that result in reproductive, developmental, or generational effects. Although a few of the toxicants considered in this report are known to be reproductive or developmental toxicants or mutagens (e.g., benzene), for none of them is there strong evidence that they affect germ cells and cause developmental effects in offspring when one parent or the other was exposed to the toxicant prior to conception (see Tables 8-1 and 8-2). What effects occur in parental germ cells, what happens after the exposure ceases, and whether and how lifecourse events compound epigenetic alterations are some of the knowledge gaps related to the nature of these alterations, as discussed below.
- Heritable transmission A critical knowledge gap continues to be the nature of the heritable factor (e.g., DNA, histone modifications, RNA) that is perturbed by specific exposures and
- responsible for generational transmission. The types of studies that can address this knowledge gap include mechanistic studies using cell-based models or human tissues (e.g., blood, semen, saliva, and urine) or multigenerational animal model studies to understand how deployment-related exposures affect DNA, histone modifications, and RNA. Other important studies are genetic/epigenetic studies using comprehensive, unbiased next-generation approaches with state-of-the-art techniques for genomic, epigenomic, chromatin conformation, and transcriptional profiling as well as proteomic and other omics technologies. Studies in model organisms on the early detection and reversibility of exposure-related epigenetic alterations and adverse health outcomes should also be encouraged, as such interventions would be very valuable if they could be developed and applied to humans to mitigate or prevent the adverse outcomes associated with deployment-related exposures.
- Persistence The committee uses the term “persistence” to refer to how long effects are maintained after the discontinuation of exposure. Alterations that remain after the exposure ceases can occur in germ cells or somatic cells in other target tissues and can result in an adverse phenotype across generations. There is a need to identify persistent alterations that are relevant (i.e., casually linked) to the generational inheritance of effects. In the case of human studies, it will be important to collect baseline or background biological samples from veterans (e.g., semen) to make sure that such alterations were not present before the exposure.
- Gene x environment (GxE) interactions In ways both subtle and obvious, the response to environmental exposures is determined not only by the nature of the exposure itself but also by the genetics and epigenetics of the exposed individuals—what is often termed gene x environment interactions. Similar interactions also occur at the level of the epigenome (epigenome x environment; ExE interactions), as allele specificity and DNA polymorphisms can modulate the vulnerability of neighboring epigenetic marks to deployment-related exposures. It will be important to undertake both GxE and ExE studies in order to understand how these interactions influence generational inheritance and also to carry out specific studies aimed at providing actionable information, including the identification of P450 and other genetic polymorphisms that modulate the risks to specific exposures and generational inheritance by altering chemical metabolism.
- Modifiers The impact of modifiers such as stress, obesity, drugs, or diet on the type or persistence of genetic and epigenetic changes. For example, it is important to understand the role that modifiers such as stress or the immune system’s response to vaccines and novel antigens play in shaping responses to deployment exposures. This should be considered as a function of age-related methylation that is known to represent developmentally programmed changes that occur throughout the genome and the accumulation of random changes as the epigenome drifts. Epigenetic drift, the gradual change away from baseline, reflects the fidelity of maintaining the epigenetic marks (Issa, 2014). Together, the age-related methylation and accumulation of random changes constitute environmental deflection that can alter the physiological outcome of the other GxE modifiers (Kochmanski et al., 2017).
As summarized in Chapter 8, the reproductive and developmental health outcomes that may be associated with parental deployment exposures include the following:
- behavioral and neurological dysfunction in offspring associated with maternal exposure to organophosphate pesticides;
- birth defects associated with maternal exposure to polycyclic aromatic hydrocarbons and glycol ethers;
- persistent respiratory diseases such as asthma associated with maternal exposure to particulate matter (PM);
- childhood leukemia associated with parental exposure to benzene; and
- adverse reproductive and pregnancy outcomes (not limited to infertility) associated with several of the agents of concern, including hexavalent chromium, PM, several pesticides, and glycols and glycol ethers.
Chapter 8 also identified several toxicants—for example, depleted uranium, solvents, and lindane—for which the animal data showed reproductive or developmental effects but few data existed to support similar findings in humans. While there is a growing human and animal evidence base on the reproductive and developmental effects of many of the toxicants of concern to exposed men and women and their children, there is still relatively little information on the specific effects of veterans’ exposures on their children, grandchildren, and great-grandchildren. Animal models will be particularly useful to begin to address these knowledge gaps. They can provide information on the biological plausibility of deployment-related health outcomes and insights into the mechanisms that underlie such effects through several different types of studies.
Transgenerational studies in animal models Generational studies that focus on meiotic inheritance will need to be done on the various exposures of concerns, and they will need to assess the impact of the generational inheritance variables discussed above (e.g., persistence, GxE interactions). Conducting multigenerational studies in animals poses a number of challenges. The typical generational animal studies required by regulatory agencies to protect human health expose animals to a toxicant throughout the entire study period, and each generation is exposed. These studies are not particularly helpful for trying to determine the types of generational effects that might manifest in offspring following parental exposure during deployment. In such cases typically the father would have been exposed before the conception of the child, or the mother would have been exposed before conception or during the early prenatal period, or perhaps both. Generational animal studies should look at parental exposure (and gestational exposure of the offspring), but the effects should also be studied across multiple generations, at least to the F2 (for paternal exposures) and F3 (for maternal exposures) generation, in order to study potential transgenerational effects among offspring which would have not had any exposure to the agent (see Figure 3-2 in Chapter 3).
It is also essential in animal studies to take sex into account as a biological variable for both parental exposures and offspring effects. Therefore, mechanistic studies on multigenerational inheritance should focus on toxicant exposures (preconceptional or affecting the parental or offspring germ cells) that yield a reproducible phenotype in the offspring or subsequent generations, coupled with an examination of alterations associated with these exposures in germ cells or in the offspring (or both) or modifications caused by the surrounding environment (i.e., lifecourse exposures).
Importantly, epigenetic alterations induced by environmental exposures may be further modified postexposure (e.g., erased or altered by subsequent environmental exposures), and often the adverse effects of these perturbations are only revealed in response to later life environmental exposures (Walker and Ho, 2012). For example, early life exposures to exogenous estrogens cause reprogramming of the prostate epigenome. In the case of androgen-responsive
genes that are reprogrammed, the effect of epigenetic changes induced by early life exogenous estrogen exposure is seen only when the reprogrammed animals are exposed to exogenous androgen later in life. In these animals, the reprogrammed androgen-responsive genes exhibit an exaggerated response to testosterone, becoming more highly expressed than those in control animals and promoting the development of prostate tumors (Wang et al. 2016). This makes clear the potential for the post-deployment environment to influence whether future generations exhibit an adverse health effect that is related to parental deployment exposures and, importantly, the possibility that adverse health effects could “skip” a generation if lifetime exposures do not act to reveal those effects. Accordingly, animal studies that examine the effects of the postexposure environment (e.g., lifestyle, diet, drugs, occupational exposures) on epigenetic reprogramming and multigenerational inheritance should be encouraged.
Also of importance is the sex-specificity of epigenetic reprogramming by environmental exposures and also of multigenerational inheritance. Elucidating the role of sex will require studies on how both maternal and paternal lineages are affected by environmental exposures, how subsequent genetic and epigenetic alterations can be passed through the germ line, and whether both male and female offspring are affected by the alterations. For example, males may be at a greater risk of neurological deficits than females following prenatal exposure to certain organophosphate pesticides (Furlong et al., 2017).
- Impact on reproductive function Examples of studies to address how deployment-related exposures may affect reproductive outcomes include studies of toxicant effects on sexual performance, research on ovulatory cycles and parturition, and the screening of semen samples beyond fertility measures to identify potential markers of reproductive and generational effects. Other studies might explore the disease processes for polycystic ovary syndrome, endometriosis, and sexual dysfunction.
- Identification of adverse phenotypes Little is known regarding how a deployment-related exposure would manifest as a generationally heritable adverse phenotype. Knowledge gaps include whether there is a correlation between exposure-related adult effects and manifestations in subsequent generations and whether the adverse effects seen in the offspring of exposed veterans are indeed caused by specific deployment-related exposures. Thus, there is a pressing need for identifying robust phenotypes for use in tracking multi- and transgenerational effects, both to facilitate model organism studies and for translation to humans. Such phenotypic assessments would also be key for obtaining evidence of persistent epigenetic alterations of gametes that are linked to adverse outcomes in offspring.
There are many data and knowledge gaps preventing a clearer understanding of how deployment exposures can affect children or descendants that can be addressed by basic research. In some cases it may be advantageous to conduct animal and mechanistic research rather than epidemiologic studies or a health monitoring program, given the ethical, time, and resource constraints inherent in human research. However, an expanded understanding of how deployment exposures can affect the health of veteran’s children or other descendants will require an integrated approach informed by health monitoring, epidemiological studies, and basic research. The committee strongly believes that such an understanding will be facilitated by consistent and multifaceted collaborations among a variety of research institutions, both governmental and nongovernmental, that seek to improve the health of veterans and their descendants.
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