10


Effects 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



The National Academies | 500 Fifth St. N.W. | Washington, D.C. 20001
Copyright © National Academy of Sciences. All rights reserved.
Terms of Use and Privacy Statement



Below are the first 10 and last 10 pages of uncorrected machine-read text (when available) of this chapter, followed by the top 30 algorithmically extracted key phrases from the chapter as a whole.
Intended to provide our own search engines and external engines with highly rich, chapter-representative searchable text on the opening pages of each chapter. Because it is UNCORRECTED material, please consider the following text as a useful but insufficient proxy for the authoritative book pages.

Do not use for reproduction, copying, pasting, or reading; exclusively for search engines.

OCR for page 726
10 Effects 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 find- ings 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 726

OCR for page 726
EFFECTS ON FUTURE GENERATIONS 727 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 chap- ter focuses and expands on issues related to possible adverse effects in future g ­ enerations—both the children of Vietnam veterans and their offspring in turn. Since its inception, the VAO series has considered birth defects (primarily lim- ited 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’ chil- dren 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 under­ ying l 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 Viet- nam 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

OCR for page 726
728 VETERANS AND AGENT ORANGE: UPDATE 2012 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 veter- ans, 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 mobiliza- tion 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 informa- tion 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, inter- nal 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.

OCR for page 726
EFFECTS ON FUTURE GENERATIONS 729 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 out- comes 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 devel- opment 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 epi- genetic modification of an individual’s somatic cell lines may not be manifested for long periods of time. In epigenetic trans­ enerational inheritance, an alteration g 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 ­ kinner, 2007), so this process requires exposure precisely at the time in germ- S 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 absorp- tion 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 re- view biologic plausibility and relevant data on female veterans and male veterans separately because the underlying pathways for adverse effects in offspring are so different.

OCR for page 726
730 VETERANS AND AGENT ORANGE: UPDATE 2012 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 veter- ans 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 pre­ conception 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 methyla­ion than somatic cells and unique DNA methylation marks (particularly t 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 possibil- ity (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 modi- fication patterns are retained and thus may transmit chemical-induced altera­ ions t 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 devel- oping 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

OCR for page 726
EFFECTS ON FUTURE GENERATIONS 731 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, concentra- tions 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 inter- course 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 ­ verage of 26 years after the possible exposure. Of those, 6 had TCDD greater a 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 vol- ume 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 measure- ments, 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 compar- ing TCDD concentrations in serum and seminal fluid. Furthermore, despite the potential for a seminal-fluid route of exposure, the

OCR for page 726
732 VETERANS AND AGENT ORANGE: UPDATE 2012 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 con- stituents 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 pater- nally 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 de- fects 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 evi- dence that convincingly demonstrated that paternal exposure to any particular chemical before conception resulted in cancer or birth defects in offspring. How- ever, there are few data for addressing the hypothesis of paternal exposure and adverse effects in human offspring in which the exposure occurred before concep- tion only to the father and was measured with an objective dosimeter. Thus, it is

OCR for page 726
EFFECTS ON FUTURE GENERATIONS 733 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. Sev- eral systematic reviews of the topic have been conducted (Chia and Shi, 2002; W ­ eselak 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 lacta- tion. Dioxin in the mother’s bloodstream can cross the placenta and expose the developing embryo and fetus. Furthermore, mobilization of dioxin during preg- nancy 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 spermato- gonia) in offspring undergo critical developmental stages during fetal life, and

OCR for page 726
734 VETERANS AND AGENT ORANGE: UPDATE 2012 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 develop- ment, 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 com- plicated by differences in sensitivity and susceptibility among individual animals, strains, and species; by differences in route, dose, duration, and timing of expo- sure 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-­richlorophenoxyacetic t 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, func- tion, or metabolism, whether genetically determined or resulting from an envi- ronmental 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 physi- cal 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, occu- pational 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 pa- ternal preconception exposures as a contributing factor to the occurrence of birth defects (Chow et al., 2009; Desrosiers et al., 2012; Dohle, 2010; Schull, 2003).

OCR for page 726
EFFECTS ON FUTURE GENERATIONS 735 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 sugges- tive 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. 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

OCR for page 726
736 VETERANS AND AGENT ORANGE: UPDATE 2012 TABLE 10-1  Selected Epidemiologic Studies—Birth Defects in Offspring of Subjectsa (Shaded Entries Are New Information for This Update) Exposure of Interest/Estimated Exposed Relative Risk Study Population Casesb (95% CI)b Reference VIETNAM VETERANS US Vietnam Veterans US Air Force Health Study—Ranch Hand All COIs veterans vs SEA veterans (unless otherwise noted) Verified birth defects in children born to Michalek et al., AFHS veterans 1998a Before service in SEA nr 0.7 (nr) After service in SEA nr 1.5 (nr) High-exposure Ranch Hands relative to Wolfe et al.,1995 comparisons All anomalies 57 1.0 (0.8–1.3) Nervous system 3 nr Eye 3 1.6 (0.4–6.0) Ear, face, neck 5 1.7 (0.6–4.7) Circulatory system, heart 4 0.9 (0.3–2.7) Respiratory system 2 nr Digestive system 5 0.8 (0.3–2.0) Genital system 6 1.2 (0.5–3.0) Urinary system 7 2.1 (0.8–5.4) Musculoskeletal 31 0.9 (0.6–1.2) Skin 3 0.5 (0.2–1.7) Chromosomal anomalies 1 nr CDC Birth Defects Study—Hospital All COIs records reviewed for offspring of 7,924 Vietnam veterans and 7,364 non–Vietnam veterans General Birth Defects Study—hospital 130 1.0 (0.8–1.3) CDC, 1989b records Major birth defects 51 1.2 (0.8–1.9) Digestive system defects 18 2.0 (0.9–4.6) Birth defects—black Vietnam veterans 21 3.4 (1.5–7.6) only Vietnam veterans identified through CDC Erikson et al., Metropolitan Atlanta Congenital Defects 1984a Program Any major birth defects 428 1.0 (0.8–1.1) Multiple birth defects with reported 25 1.1 (0.7–1.7) exposure EOI-5: spina bifida 1 2.7 (1.2–6.2) EOI-5: cleft lip with or without cleft 5 2.2 (1.0–4.9) palate

OCR for page 726
762 VETERANS AND AGENT ORANGE: UPDATE 2012 female offspring (Ding et al., 2011; McConaha et al., 2011). Exposure of gestat- ing 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 ap- peared 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 genera- tions F1–F3. Another mode of epigenetic change is modification of the spatial arrange- ment of chromosomes, which can influence gene expression and cell differentia- tion. 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 influ- ence 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 neu- rologic, 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 expo- sures before conception of the next generation, there is insufficient evidence of transgenerational affects.

OCR for page 726
EFFECTS ON FUTURE GENERATIONS 763 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 labora- tory 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. REFERENCES1 ACS (American Cancer Society). 2010. Cancer Facts and Figures 2010. http://www.cancer.org/acs/ groups/content/@nho/documents/document/acspc–024113.pdf (accessed May 16, 2011). ACS. 2013. Cancer Facts and Figures 2013. Atlanta, GA: American Cancer Society. ADVA (Australia Department of Veterans Affairs). 1983. Case-Control Study of Congenital Anoma- lies and Vietnam Service. Canberra, Australia: ADVA. Ahmed RG. 2011. Perinatal TCDD exposure alters developmental neuroendocrine system. Food and Chemical Toxicology 49:1276–1284. AIHW (Australian Institute of Health and Welfare). 1999. Morbidity of Vietnam Veterans: A Study of the Health of Australia’s Vietnam Veteran Community: Volume 3: Validation Study. Canberra, Australia. AIHW. 2000. Morbidity of Vietnam Veterans. Adrenal Gland Cancer, Leukaemia and Non-Hodgkin’s Lymphoma: Supplementary Report No. 2. (AIHW cat. no. PHE 28). Canberra, Australia: AIHW. AIHW. 2001. Morbidity of Vietnam Veterans. Adrenal Gland Cancer, Leukaemia and Non-Hodgkin’s Lymphoma: Supplementary Report No. 2. Revised edition (AIHW cat. No. PHE 34). Canberra, Australia: AIHW. Aschengrau A, Monson RR. 1990. Paternal military service in Vietnam and the risk of late adverse pregnancy outcomes. American Journal of Public Health 80(10):1218–1224. Barker DJP, Lampl M, Roseboom T, Winder N. 2012. Resource allocation in utero and health in later life. Placenta 33:e30–e34. Bertazzi PA, Zocchetti C, Pesatori AC, Guercilena S, Consonni D, Tironi A, Landi MT. 1992. Mortality of a young population after accidental exposure to 2,3,7,8-tetrachlorodibenzodioxin. International Journal of Epidemiology 21(1):118–123. Blatter BM, Hermens R, Bakker M, Roeleveld N, Verbeek AL, Zielhuis GA. 1997. Paternal occupa- tional exposure around conception and spina bifida in offspring. American Journal of Industrial Medicine 32(3):283–291. Bloom AD, ed. 1981. Guidelines for Studies of Human Populations Exposed to Mutagenic and Re- productive Hazards. White Plains, NY: March of Dimes Foundation. Brown NM, Manzolillo PA, Zhang JX, Wang J, Lamartiniere CA. 1998. Prenatal TCDD and predis- position to mammary cancer in the rat. Carcinogenesis 19(9):1623–1629. Bruner-Tran KL, Osteen KG. 2011. Developmental exposure to TCDD reduces fertility and negatively affects pregnancy outcomes across multiple generations. Reproductive Toxicology 31:344–350. 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.

OCR for page 726
764 VETERANS AND AGENT ORANGE: UPDATE 2012 Buckley JD, Robison LL, Swotinsky R, Garabrant DH, LeBeau M, Manchester P, Nesbit ME, Odom L, Peters JM, Woods WG, Hammond GD. 1989. Occupational exposures of parents of children with acute nonlymphocytic leukemia: A report from the Childrens’ Cancer Study Group. Cancer Research 49(14):4030–4037. CDC (Centers for Disease Control and Prevention). 1989a. Health Status of Vietnam Veterans. Vietnam Experience Study, Vol. V, Reproductive Outcomes and Child Health. Atlanta, GA: US Department of Health and Human Services. CDC. 1989b. Health Status of Vietnam Veterans. Vietnam Experience Study, Vol. V, Reproductive Outcomes and Child Health. Atlanta, GA: US Department of Health and Human Services. Chao HR, Wang SL, Lee CC, Yu HY, Lu YK, Päpke O. 2004. Level of polychlorinated dibenzo-p- dioxins, dibenzofurans and biphenyls (PCDD/Fs, PCBs) in human milk and the input to infant body burden. Food and Chemical Toxicology 42:1299–1308. Chapin RE, Robbins WA, Schieve LA, Sweeney AM, Tabacova SA, Tomashek KM. 2004. Off to a good start: The influence of pre- and periconceptional exposures, parental fertility, and nutrition on children’s health. Environmental Health Perspectives 112(1):69–78. Chen Z, Stewart PA, Davies S, Giller R, Krailo M, Davis M, Robison L, Shu XO. 2005. Parental occupational exposure to pesticides and childhood germ-cell tumors. American Journal of Epidemiology 162(9):858–867. Chen Z, Robison L, Giller R, Krailo M, Davis M, Davies S, Shu XO. 2006. Environmental exposure to residential pesticides, chemicals, dusts, fumes, and metals, and risk of childhood germ cell tumors. International Journal of Hygiene and Environmental Health 209(1):31–40. Chia SE, Shi LM. 2002. Review of recent epidemiological studies on paternal occupations and birth defects. Occupational and Environmental Medicine 59(3):149–155. Chokkalingam AP, Metayer C, Scelo GA, Chang JS, Urayama KY, Aldrich MC, Guha N, Hansen HM, Dahl GV, Barcellos LF, Wiencke JK, Wiemels JL, Buffler PA. 2012. Variation in xenobiotic transport and metabolism genes, household chemical exposures, and risk of childhood acute lymphoblastic leukemia. Cancer Causes and Control 23(8):1367–1375. Chow EJ, Kamineni A, Daling, JR, Fraser A, Wiggins CL, Mineau GP, Hamre MR, Severson RK, Drews-Botsch C, Mueller BA. 2009. Reproductive outcomes in male cancer survivors: A linked cancer-birth registry analysis. Archives of Pediatric and Adolescent Medicine 163(10):887–894. Codrington AM, Hales BF, Robaire B. 2004. Spermiogenic germ cell phase-specific DNA damage following cyclophosphamide exposure. Journal of Andrology 25(3):354–362. Cooney MA, Daniels JL, Ross JA, Breslow NE, Pollock BH, Olshan AF. 2007. Household pesticides and the risk of Wilms tumor. Environmental Health Perspectives 115(1):134–137. Cordier S, Chevrier C, Robert-Gnansia E, Lorente C, Brula P, Hours M. 2004. Risk of congenital anomalies in the vicinity of municipal solid waste incinerators. Occupational and Environmental Medicine 61(1):8–15. Cordier S, Lehebel A, Amar E, Anzivino-Viricel L, Hours M, Monfort C, Chevrier C, Chiron M, Robert-Gnansia E. 2010. Maternal residence near municipal waste incinerators and the risk of urinary tract birth defects. Occupational and Environmental Medicine 67(7):493–499. Daniels JL, Olshan AF, Teschke K, Herz-Picciotto I, Savitz DA, Blatt J, Bondy ML, Neglia JP, ­ ollock P BH, Cohn SL, Look AT, Seeger RC, Castleberry RP. 2001. Residential pesticide exposure and neuroblastoma. Epidemiology 12:20–27. De Roos AJ, Olshan AF, Teschke K, Poole C, Savitz DA, Blatt J, Bondy ML, Pollock BH. 2001a. Parental occupational exposures to chemicals and incidence of neuroblastoma in offspring. American Journal of Epidemiology 154(2):106–114. De Roos AJ, Teschke K, Savitz DA, Poole C, Grufferman S, Pollock BH, Olshan AF. 2001b. Parental occupational exposures to electromagnetic fields and radiation and incidence of neuroblastoma in offspring. Epidemiology 12(5):508–517.

OCR for page 726
EFFECTS ON FUTURE GENERATIONS 765 Desaulniers D, Leingartner K, Musicki B, Cole J, Li M, Charboneau M, Tsang BK. 2004. Lack of effects of postnatal exposure to a mixture of aryl hydrocarbon-receptor agonists on the devel- opment of methylnitrosourea-induced mammary tumors in Sprague-Dawley rats. Journal of Toxicology and Environmental Health Part A 67(18):1457–1475. Desrosiers TA, Herring AH, Shapira SK, Hooiveld M, Luben TJ, Herdt-Losavio ML, Olshan AF; National Birth Defects Prevention Study. 2012. Paternal occupation and birth defects: Findings from the National Birth Defects Prevention Study. Occupational and Environmental Medicine 69(8):534–542. Dimich-Ward H, Hertzman C, Teschke K, Hershler R, Marion SA, Ostry A, Kelly S. 1996. Reproduc- tive effects of paternal exposure to chlorophenate wood preservatives in the sawmill industry. Scandinavian Journal of Work, Environment and Health 22(4):267–273. Ding T, McConaha M, Boyd KL, Osteen KG, Bruner-Tran KL. 2011. Developmental dioxin exposure of either parent is associated with an increased risk of preterm birth in adult mice. Reproductive Toxicology 31:351–358. Dohle GR. 2010. Male infertility in cancer patients: Review of the literature. International Journal of Urology 17:327–331. Dong B, Nishimura N, Vogel CF, Tohyama C, Matsumura F. 2010. TCDD-induced cyclooxygenase-2 expression is mediated by the nongenomic pathway in mouse MMDD1 macula densa cells and kidneys. Biochemical Pharmacology 79(3):487–497. Donovan JW, MacLennan R, Adena M. 1984. Vietnam service and the risk of cogenital anomalies: A case-control study. Medical Journal of Australia 140(7):394–397. Dragin N, Dalton TP, Miller ML, Shertzer HG, Nebert DW. 2006. For dioxin-induced birth defects, mouse or human CYP1A2 in maternal liver protects whereas mouse CYP1A1 and CYP1b1 are inconsequential. Journal of Biological Chemistry 281(27):18591–18600. Draper GJ, Little MP, Sorahan T, Kinlen LJ, Bunch KJ, Conquest AJ, Kendall GM, Kneale GW, L ­ ancashire RJ, Muirhead CR, O’Connor CM, Vincent TJ. 1997. Cancer in the offspring of radia- tion workers: A record linkage study. British Medical Journal 315(7117):1181–1188. Erickson JD, Mulinare J, Mcclain P, Fitch T, James L, McClearn A, Adams M. 1984a. Vietnam Vet- erans’ Risks for Fathering Babies with Birth Defects. Atlanta, GA: US Department of Health and Human Services, Centers for Disease Control. Erickson JD, Mulinare J, McClain PW, Fitch TG, James LM, McClearn AB, Adams MJ. 1984b. Vietnam veterans’ risks for fathering babies with birth defects. Journal of the American Medical Association 252(7):903–912. Falahatpisheh MH, Nanez A, Ramos KS. 2011. AHR regulates WT1 genetic programming during murine nephrogenesis. Molecular Medicine 17(11–12):1275–1284. Fear NT, Hey HK, Vincent T, Murphy M. 2007. Paternal occupation and neural tube defects: A case- control study based on the Oxford Record Linkage Study register. Paediatric and Perinatal Epidemiology 21(2):163–168. Fenton SE, Hamm JT, Birnbaum LS, Youngblood GL. 2000. Adverse effects of TCDD on mammary gland development in Long Evans rats: A two generational study. Organohalogen Compounds 48:157–160. Fenton SE, Hamm JT, Birnbaum LS, Youngblood GL. 2002. Persistent abnormalities in the rat mammary gland following gestational and lactational exposure to 2,3,7,8–tetrachlorodibenzo- p-dioxin (TCDD). Toxicological Sciences 67(1):63–74. Fernández M, Paradisi M, D’Intino G, Del Vecchio G, Sivilia S, Giardino L, Calzà L. 2010. A single prenatal exposure to the endocrine disruptor 2,3,7,8-tetrachlorodibenzo-p-dioxin alters devel- opmental myelination and remyelination potential in the rat brain. Journal of Neurochemistry 115(4):897–909. Field B, Kerr C. 1988. Reproductive behaviour and consistent patterns of abnormality in offspring of Vietnam veterans. Journal of Medical Genetics 25:819–826.

OCR for page 726
766 VETERANS AND AGENT ORANGE: UPDATE 2012 Fitzgerald EF, Weinstein AL, Youngblood LG, Standfast SJ, Melius JM. 1989. Health effects three years after potential exposure to the toxic contaminants of an electrical transformer fire. Archives of Environmental Health 44:214–221. Flower KB, Hoppin JA, Lynch CF, Blair A, Knott C, Shore DL, Sandler DP. 2004. Cancer risk and parental pesticide application in children of Agricultural Health Study participants. Environmen- tal Health Perspectives 112(5):631–635. Foster WG, Maharaj-Briceño S, Cyr DG. 2011. Dioxin-induced changes in epididymal sperm count and spermatogenesis. Ciência and Saúde Coletiva 16(6):2893–2905. García AM, Benavides FG, Fletcher T, Orts E. 1998. Paternal exposure to pesticides and congenital malformations. Scandinavian Journal of Work, Environment and Health 24(6):473–480. Gardner MJ, Snee MP, Hall AJ, Powell CA, Downes S, Terrell JD. 1990. Results of case-control study of leukaemia and lymphoma among young people near Sellafield nuclear plant in West Cumbria. British Medical Journal 300(6722):423–429. Garry VF, Schreinemachers D, Harkins ME, Griffith J. 1996. Pesticide appliers, biocides, and birth defects in rural Minnesota. Environmental Health Perspectives 104(4):394–399. Gassmann K, Abel J, Bothe H, Haarmann-Stemmann T, Merk HF, Quasthoff KN, Rockel TD, S ­ chreiber T, Fritsche E. 2010. Species-specific differential AHR expression protects human neural progenitor cells against developmental neurotoxicity of PAHs. Environmental Health Perspectives 118(11):1571–1577. Green DM, Kawashima T, Stovall M, Leisenring W, Sklar CA, Mertens AC, Donaldson SS, Byrne J, Robison LL. 2010. Fertility of male surviviors of childhood cancer: A report from the Childhood Cancer Survivor Study. Journal of Clinical Oncology 28(2):332–339. Hales BF, Grenier L, Lalancette C, Robaire B. 2011. Epigenetic programming: From gametes to blasatocyst. Birth Defects Research (Part A) 91:652–665. Hamatani T. 2012. Human spermatozoal RNAs. Fertility and Sterility (9792):275–281. Hammoud SS, Nix DA, Zhang H, Purwar J, Carrell DT, Cairns BR. 2009. Distinctive chromatin in human sperm packages genes for embryo development. Nature 460:473–478. Hanify JA, Metcalf P, Nobbs CL, Worsley KJ. 1981. Aerial spraying of 2,4,5-T and human birth malformations: An epidemiological investigation. Science 212:349–351. Heacock H, Hogg R, Marion SA, Hershler R, Teschke K, Dimich-Ward H, Demers P, Kelly S, Ostry A, Hertzman C. 1998. Fertility among a cohort of male sawmill workers exposed to chloro- phenate fungicides. Epidemiology 9(1):56–60. Holladay SD, Mustafa A, Gogal RM Jr. 2011. Prenatal TCDD in mice increases adult autoimmunity. Reproductive Toxicology 31:312–318. Hou L, Zhang X, Wang D, Baccarelli A. 2011. Environmental chemical exposures and human epi- genetics. International Journal of Epidemiology 41(1):79–105. Howell SJ, Shalet SM. 2005. Spermatogenesis after cancer treatment: Damage and recovery. Journal of the National Cancer Institute Monographs 34:12–17. Humblet O, Williams PL, Korrick SA, Sergeyev O, Emond C, Birnbaum LS, Burns JS, Altshul L, Patterson DG Jr, Turner WE, Lee MM, Revich B, Hauser R. 2011. Dioxin and polychlorinated biphenyl concentrations in mother’s serum and the timing of pubertal onset in sons. Epidemiol- ogy 22(6):827–835. Infante-Rivard C, Labuda D, Krajinovic M, Sinnett D. 1999. Risk of childhood leukemia associated with exposure to pesticides and with gene polymorphisms. Epidemiology 10:481–487. IOM (Institute of Medicine). 1994. Veterans and Agent Orange: Health Effects of Herbicides Used in Vietnam. Washington DC: National Academy Press. IOM. 1996. Veterans and Agent Orange: Update 1996. Washington, DC: National Academy Press. IOM. 1999. Veterans and Agent Orange: Update 1998. Washington, DC: National Academy Press. IOM. 2001. Veterans and Agent Orange: Update 2000. Washington, DC: National Academy Press. IOM. 2002. Veterans and Agent Orange: Herbicide/Dioxin Exposure and Acute Myelogenous Leuke- mia in the Children of Vietnam Veterans. Washington, DC: National Academy Press.

OCR for page 726
EFFECTS ON FUTURE GENERATIONS 767 IOM. 2003. Veterans and Agent Orange: Update 2002. Washington, DC: The National Academies Press. IOM. 2005. Veterans and Agent Orange: Update 2004. Washington, DC: The National Academies Press. IOM. 2007. Veterans and Agent Orange: Update 2006. Washington, DC: The National Academies Press. IOM. 2009. Veterans and Agent Orange: Update 2008. Washington, DC: The National Academies Press. IOM. 2011. Veterans and Agent Orange: Update 2010. Washington, DC: The National Academies Press. Izumi S, Koyama K, Soda M, Suyama A. 2003. Cancer incidence in children and young adults did not increase relative to parental exposure to atomic bombs. British Journal of Cancer 89:1709–1713. Jacobs H, Dennefeld C, Féret B, Viluksela M, Håkansson H, Mark M, Ghyselinck NB. 2011. Retinoic acid drives aryl hydrocarbon receptor expression and is instrumental to dioxin-induced toxicity during palate development. Environmental Health Perspectives 119(11):1590–1595. Jang JY, Shin S, Choi BI, Park D, Jeon JH, Hwang SY, Kim JC, Kim YB, Nahm SS. 2007. Antiterato- genic effects of alpha-naphthoflavone on 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) exposed mice in utero. Reproductive Toxicology 24(3-4):303–309. Jang JY, Park D, Shin S, Jeon JH, Choi Bi, Joo SS, Hwang SY, Nahm SS, Kim YB. 2008. Antitera- togenic effect of resveratrol in mice exposed in utero to 2,3,7,8-tetrachlorodibenzo-p-dioxin. European Journal of Pharmacology 591(1–3):280–283. Jenkins TG, Carrell DT. 2012a. Dynamic alterations in the paternal epigenetic landscape following fertilization. Frontiers in Genetics 3:1–8. Jenkins TG, Carrell DT. 2012b. The sperm epigenome and potential implications for the developing embryo. Reproduction 143:727–734. Jirtle RL, Skinner MS. 2007. Environmental epigenomics and disease susceptibility. Nature Reviews Genetics 8:253–262. Jusko TA, De Roos AJ, Schwartz SM, Lawrence BP, Palkovicova L, Nemessanyi T, Drobna B, F ­ abisikova A, Kocan A, Jahnova E, Kavanagh TJ, Trnovec T, Hertz-Picciotto I. 2011. Maternal and early postnatal polychlorinated biphenyl exposure in relation to total serum immunoglobulin concentrations in 6-month-old infants. Journal of Immunotoxicology 8(1):95–100. Kalter H, Warkany J. 1983. Congenital malformations. Etiologic factors and their role in prevention (first of two parts). New England Journal of Medicine 308:424–431. Kang HK, Mahan CM, Lee KY, Magee CA, Mather SH, Matanoski G. 2000. Pregnancy outcomes among US women Vietnam veterans. American Journal of Industrial Medicine 38(4):447–454. Kawano M, Kawaji H, Grandjean V, Klani J, Rassoulzadegan M. 2012. Novel small noncoding RNAs in mouse spermatozo, zygotes and early embryos. PLOS ONE 7(9):e44542. Kerr M, Nasca PC, Mundt KA, Michalek AM, Baptiste MS, Mahoney MC. 2000. Parental occupa- tional exposures and risk of neuroblastoma: A case-control study (United States). Cancer Causes and Control 11:635–643. Kinlen LJ. 1993. Can paternal preconceptional radiation account for the increase of leukaemia and non-Hodgkin’s lymphoma in Seacale. British Medical Journal 306(6894):1718–1721. Kinlen LJ, Clarke K, Balkwaill A. 1993. Paternal preconceptional radiation exposure in the nuclear industry and leukaemia and non-Hodgkin’s lymphoma in young people in Scotland. British Medical Journal 306(6886):1153–1158. Klemmt L, Scialli AR. 2005. The transport of chemicals in semen. Birth Defects Research (Part B) 74:119–131. Kramer JA, Krawetz SA. 1997. RNA in spermatozoa: Implications for the alternative haploid genome. Molecular Human Reproduction 3(6):473­–478.

OCR for page 726
768 VETERANS AND AGENT ORANGE: UPDATE 2012 Krawetz SA. 2005. Paternal contribution: New insights and future challenges. Nature Reviews Genetics 6:633–642. Krawetz SA, Kruger A, Lalancette C, Tagett R, Anton E, Draghici S, Diamond MP. 2011. A survey of small RNAs in human sperm. Human Reproduction 26(12):3401–3412. Kristensen P, Andersen A, Irgens LM, Bye AS, Sundheim L. 1996. Cancer in offspring of parents engaged in agricultural activities in Norway: Incidence and risk factors in the farm environment. International Journal of Cancer 65(1):39–50. Kristensen P, Irgens LM, Andersen A, Bye AS, Sundheim L. 1997. Birth defects among offspring of Norwegian farmers, 1967–1991. Epidemiology 8(5):537–544. Kuscu OO, Caglar E, Aslan S, Durmusoglu E, Karademir A, Sandalli N. 2009. The prevalence of molar incisor hypomineralization (MIH) in a group of children in a highly polluted urban region and a windfarm–green energy island. International Journal of Paediatric Dentistry 19(3):176–185. Krysiak-Baltyn K, Toppari J, Skakkebaek NE, Jensen TS, Virtanen HE, Schramm KW, Shen H, Vartiainen T, Kiviranta H, Taboureau O, Audouze K, Brunak S, Main KM. 2012. Association between chemical pattern in breast milk and congenital cryptorchidism: Modelling of complex human exposures. International Journal of Andrology 35:294–302. Laisi S, Kiviranta H, Lukinmaa PL, Vartiainen T, Alaluusua S. 2008. Molar–incisor–­ ypomineralisation h and dioxins: New findings. European Archives of Paediatric Dentistry: Official Journal of the European Academy of Paediatric Dentistry 9(4):224–227. Lamb JC 4th, Moore JA, Marks TA, Haseman JK. 1981. Development and viability of offspring of male mice treated with chlorinated phenoxy acids and 2,3,7,8-tetrachlorodibenzo-p-dioxin. Journal of Toxicology and Environmental Health 8(5-6):835–844. Lanham KA, Peterson RE, Heideman W. 2012. Sensitivity to dioxin decreases as zebrafish mature. Toxicological Sciences 127(2):360–370. La Rocca C, Alivernini S, Badiali M, Cornoldi A, Iacovella N, Silvestroni L, Spera G, Turrio-Bal- dassarri L. 2008. TEQs and body burden for PCDDs, PCDFs, and dioxin-like PCBs in human adipose tissue. Chemosphere 73(1):92–96. Latchney SE, Lioy DT, Henry EC, Gasiewicz TA, Strathmann FG, Mayer-Pröschel M, Opanashuk LA. 2011. Neural precursor cell proliferation is disrupted through activation of the aryl hydrocarbon receptor by 2,3,7,8-tetrachlorodibenzo-p-dioxin. Stem Cells and Development 20(2):313–326. Lawson CC, Schnorr TM, Whelan EA, Deddens JA, Dankovic DA, Piacitelli LA, Sweeney MH, Connally LB. 2004. Paternal occupational exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin and birth outcomes of offspring: Birth weight, preterm delivery, and birth defects. Environmental Health Perspectives 112(14):1403–1408. Leijs MM, ten Tusscher GW, Olie K, van Teunenbroek T, van Aalderen WM, de Voogt P, Vulsma T, Bartonova A, Krayer von Krauss M, Mosoiu C, Riojas-Rodriguez H, Calamandrei G, Koppe JG. 2012. Thyroid hormone metabolism and environmental chemical exposure. Environmental Health 11(Suppl 1):S1–S10. Loffredo CA, Silbergeld EK, Ferencz C, Zhang J. 2001. Association of transposition of the great arteries in infants with maternal exposures to herbicides and rodenticides. American Journal of Epidemiology 153(6):529–536. Lorber M, Phillips L. 2002. Infant exposure to dioxin-like compounds in breast milk. Environmental Health Perspectives 110(6):A325–A332. Madanat-Harjuoja LS, Malila N, Lahteenmaki P, Pukkula E, Mulviihill JJ, Boise JD Jr, Sankila R. 2010. Risk of cancer among children of cancer patients: A nationwide study in Finland. Inter- national Journal of Cancer 126:1196–1205. Manikkam M, Guerrero-Bosagna C, Tracey R, Haque MM, Skinner MK. 2012a. Transgenerational actions of environmental compounds on reproductive disease and identification of epigenetic biomarkers of ancestral exposures. PLoS ONE 7(2):12 pps.

OCR for page 726
EFFECTS ON FUTURE GENERATIONS 769 Manikkam M, Tracey R, Guerrero-Bosagna C, Skinner MK. 2012b. Dioxin (TCDD) induces epi­ genetic transgenerational inheritance of adult onset disease and sperm epimutations. PLoS ONE 7(9):15 pps. Mastroiacovo P, Spagnolo A, Marni E, Meazza L, Betrollini R, Segni G, Brogna-Pignatti C. 1988. Birth defects in Seveso area after TCDD contamination. Journal of the American Medical Asso­ ciation 259:1668–1672 (published erratum appears in JAMA 1988, 260:792). McConaha ME, Ding T, Lucas JA, Arosh JA, Osteen KG, Bruner-Tran KL. 2011. Preconcep- tion omega-3 fatty acid supplementation of adult male mice with a history of developmental 2,3,7,8-tetrachlorodibenzo-p-dioxin exposure prevents preterm birth in unexposed female part- ners. Reproduction 142:235–241. Meinert R, Schüz J, Kaletsch U, Kaatsch P, Michaelis J. 2000. Leukemia and non-Hodgkin’s lym- phoma in childhood and exposure to pesticides: Results of a register-based case-control study in Germany. American Journal of Epidemiology 151(7):639–646. Meyer KJ, Reif JS, Veeramachaneni DN, Luben TJ, Mosley BS, Nuckols JR. 2006. Agricultural pesticide use and hypospadias in eastern Arkansas. Environmental Health Perspectives 114(10):1589–1595. Michalek JE, Albanese RA, Wolfe WH. 1998a. Project Ranch Hand II: An Epidemiologic Investiga- tion of Health Effects in Air Force Personnel Following Exposure to Herbicides—­ eproductive R Outcome Update. US Department of Commerce, National Technical Information Service. Report number AFRL-HE-BR-TR-1998-0073. Mimura J, Yamashita K, Nakamura K, Morita M, Takagi TN, Nakao K, Ema M, Sogawa K, Yasuda M, Katsuki M, Fujii-Kuriyama Y. 1997. Loss of teratogenic response to 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD) in mice lacking the Ah (dioxin) receptor. Genes to Cells 2(10):645–654. Mitsuhashi T, Yonemotob J, Soneb H, Kosugea Y, Kosakia K, Takahashi T. 2010. In utero exposure to dioxin causes neocortical dysgenesis through the actions of p27Kip1. Proceedings of the National Academy of Sciences of the United States of America 107(37):16331–16335. Miyashita C, Sasaki S, Saijo Y, Washino N, Okada E, Kobayashi S, Konishi K, Kajiwara J, Todaka T, Kishi R. 2011. Effects of prenatal exposure to dioxin-like compounds on allergies and infections during infancy. Environmental Research 111:551–558. Mocarelli P, Gerthoux PM, Needham LL, Patterson DG Jr, Limonta G, Falbo R, Signorini S, Bertona M, Crespi C, Sarto C, Scott PK, Turner WE, Brambilla P. 2011. Perinatal exposure to low doses of dioxin can permanently impair human semen quality. Environmental Health Perspectives 119(5):713–718. Monge P, Wesseling C, Guardado J, Lundberg I, Ahlbom A, Cantor KP, Weiderpass E, Partanen T. 2007. Parental occupational exposure to pesticides and the risk of childhood leukemia in Costa Rica. Scandinavian Journal of Work, Environment and Health 33(4):293–303. Moses M, Lilis R, Crow KD, Thornton J, Fischbein A, Anderson HA, Selikoff IJ. 1984. Health status of workers with past exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin in the manufacture of 2,4,5-trichlorophenoxyacetic acid: Comparison of findings with and without chloracne. Ameri- can Journal of Industrial Medicine 5(3):161–182. Mustafa A, Holladay S, Witonsky S, Zimmerman K, Manari A, Countermarsh S, Karpuzoglu E, Gogal R. 2011. Prenatal TCDD causes persistent modulation of the postnatal immune response, and exacerbates inflammatory disease, in 36-week-old lupus-like autoimmune SNF1 mice. Birth Defects Research (Part B) 92:82–94. Nagayama J, Okamura K, Iida T, Hirakawa H, Matsueda T, Tsuji H, Hasegawa M, Sato K, Ma HY, Yanagawa T, Igarashi H, Fukushige J, Watanabe T. 1998. Postnatal exposure to chlorinated dioxins and related chemicals on thyroid hormone status in Japanese breast-fed infants. Che- mosphere 37(9-12):1789–1793. NCI (National Cancer Institute). 2001. Surveillance, Epidemiology, and End Results (SEER) data- base. http://seer.cancer.gov/ScientificSystems/CanQues (accessed March 19).

OCR for page 726
770 VETERANS AND AGENT ORANGE: UPDATE 2012 Neri T, Merico V, Fiordaliso F, Salio M, Rebuzzini P, Sacchi L, Bellazzi R, Redi CA, Zuccotti M, Garagna S. 2011. The differentiation of cardiomyocytes from mouse embryonic stem cells is altered by dioxin. Toxicology Letters 202:226–236. Nishijo M, Tai PT, Nakagawa H, Maruzeni S, Anh NT, Luong HV, Anh TH, Honda R, Morikawa Y, Kido T, Nishijo H. 2012. Impact of perinatal dioxin exposure on infant growth: A cross-sectional and longitudinal studies in dioxin-contaminated areas in Vietnam. PLoS ONE 7(7):10 pps. Oikawa K, Yoshida K, Takanashi M, Tanabe H, Kiyuna T, Ogura M, Saito A, Umezawa A, Kuroda M. 2008. Dioxin interferes in chromosomal positioning through the aryl hydrocarbon receptor. Biochemical and Biophysical Research Communications 374(2):361–364. Ouko LA, Shantikumar K, Knezovich J, Haycock P, Schnugh DJ, Ramsay M. 2009. Effect of alcohol consumption on CpG methylation in the differentially methylated regions of H19 and IG-DMR in male gametes: Implications for fetal alcohol spectrum disorders. Alcoholism: Clinical and Experimental Research 33(9):1615–1627. Parker L, Craft AW, Smith J, Dickinson H, Wakeford R, Binks K, McElvenny D, Scott L, Slovak A. 1993. Geographical distribution of preconceptional radiation doses to fathers employed at the Sellafield nuclear installation, West Cumbria. British Medical Journal 307:966–971. Pearce MS, Parker L. 2000. Paternal employment in agriculture and childhood kidney cancer. Pedi- atric Hematology and Oncology 17(3):223–230. Pesatori AC, Consonni D, Tironi A, Zocchetti C, Fini A, Bertazzi PA. 1993. Cancer in a young popu- lation in a dioxin-contaminated area. International Journal of Epidemiology 22(6):1010–1013. Prescott SL. 2011. The influence of early environmental exposures on immune development and subsequent risk of allergic disease. Allergy 66(Suppl 95):4–6. Puga A. 2011. Perspectives on the potential involvement of the AH receptor-dioxin axis in cardiovas- cular disease. Toxicological Sciences 120(2):256–261. Puri D, Dhawan J, Mishra RK. 2010. The paternal hidden agenda: Epigenetic inheritance through sperm chromatin. Epigenetics 5(5):386–391. Quintanilla-Vega B, Hoover DJ, Bal W, Silbergeld EK, Waalkes MP, Anderson LD. 2000. Lead in- teraction with human protamine (HP2) as a mechanism of male reproductive toxicity. Chemical Research in Toxicology 13:594–600. Ray SS, Swanson HI. 2004. Dioxin-induced immortalization of normal human keratinocytes and silencing of p53 and p16INK4a. Journal of Biological Chemistry 279(26):27187–27193. Ren A, Qiu X, Jin L, Ma J, Li Z, Zhang L, Zhu H, Finnell RH, Zhu T. 2011. Association of selected persistent organic pollutants in the placenta with the risk of neural tube defects. Proceedings of the National Academy of Sciences of the United States of America 108(31):12770–12775. Revich B, Aksel E, Ushakova T, Ivanova I, Zuchenko N, Lyuev N, Brodsky B, Sotsov Y. 2001. Dioxin exposure and public health in Chapaevsk, Russia. Chemosphere 43(4-7):951–966. Reynolds P, Von Behren J, Gunier RB, Goldberg DE, Harnly M, Hertz A. 2005b. Agricultural pesti- cide use and childhood cancer in California. Epidemiology 16(1):93–100. Rocheleau CM, Romitti PA, Sanderson WT, Sun L, Lawson CC, Waters MA, Stewart PA, Olney RS, Reefhuis J. 2011. Maternal occupational pesticide exposure and risk of hypospadias in the National Birth Defects Prevention Study. Birth Defects Research (Part A) 91(11):927–936. Rudant J, Menegaux F, Leverger G, Baruchel A, Nelken B, Bertrand Y, Patte C, Pacquement H, Verite C, Robert A, Michel G, Margueritte G, Gandemer V, Hemon D, Clavel J. 2007. Household exposure to pesticides and risk of childhood hematopoietic malignancies: The ESCALE study (SFCE). Environmental Health Perspectives 115(12):1787–1793. Schecter A, McGee H, Stanley JS, Boggess K, Brandt-Rauf P. 1996. Dioxins and dioxin-like chemi- cals in blood and semen of American Vietnam veterans from the state of Michigan. American Journal of Industrial Medicine 30(6):647–654. Schlebusch H, Wagner U, van der Ven H, al-Hasani S, Diedrich K, Krebs D. 1989. Polychlorinated biphenyls: The occurrence of the main congeners in follicular and sperm fluids. Journal of Clinical Chemistry and Clinical Biochemistry 27(9):663–667.

OCR for page 726
EFFECTS ON FUTURE GENERATIONS 771 Schreinemachers DM. 2003. Birth malformations and other adverse perinatal outcomes in four US wheat-producing states. Environmental Health Perspectives 111(9):1259–1264. Schull WJ. 2003. The children of atomic bomb survivors. A synopsis. Journal of Radiological Protec- tion 23(4):369–384. Shaw GM, Nelson V, Olshan AF. 2002. Paternal occupational group and risk of offspring with neural tube defects. Paediatric and Perinatal Epidemiology 16(4):328–333. Skinner MK, Manikkam M, Guerrero-Bosagna C. 2010. Epigenetic transgenerational actions of en- vironmental factors in disease etiology. Trends Endocrinology and Metabolism 21(4):214–222. Slater ME, Linabery AM, Spector LG, Johnson KJ, Hilden JM, Heerema NA, Robison LL, Ross JA. 2011. Maternal exposure to household chemicals and risk of infant leukemia: A report from the Children’s Oncology Group. Cancer Causes and Control 22(8):1197–1204. Smith AH, Fisher DO, Pearce N, Chapman CJ. 1982. Congenital defects and miscarriages among New Zealand 2,4,5-T sprayers. Archives of Environmental Health 37:197–200. Smith MT, McHale CM, Wiemels JL, Zhang L, Wiencke JK, Zheng S, Gunn L, Skibola CF, Ma X, Buffler PA. 2005. Molecular biomarkers for the study of childhood leukemia. Toxicology and Applied Pharmacology 206(2):237–245. Stachel B, Dougherty RC, Lahl U, Schlösser M, Zeschmar B. 1989. Toxic environmental chemicals in human semen: Analytical method and case studies. Andrologia 21(3):282–291. Stockbauer JW, Hoffman RE, Schramm WF, Edmonds LD. 1988. Reproductive outcomes of mothers with potential exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin. American Journal of Epidemiol- ogy 128:410–419. Su PH, Huang PC, Lin CY, Ying TH, Chen JY, Wang SL. 2012. The effect of in utero exposure to dioxins and polychlorinated biphenyls on reproductive development in eight year-old children. Environment International 39:181–187. Suh N, Blelloch R. 2011. Small RNAs in early mammalian development: From gametes to gastrula- tion. Development 138:1653–1661. Suskind RR, Hertzberg VS. 1984. Human health effects of 2,4,5-T and its toxic contaminants. Journal of the American Medical Association 251:2372–2380. Suzuki G, Nakano M, Nakano S. 2005. Distribution of PCDDs/PCDFs and co-PCBs in human maternal blood, cord blood, placenta, milk, and adipose tissue: Dioxins showing high toxic equivalency factor accumulate in the placenta. Bioscience, Biotechnology and Biochemistry 69(10):1836–1847. Tait S, La Rocca C, Mantovani A. 2011. Exposure of human fetal penile cells to different PCB mixtures: Transcriptome analysis points to diverse modes of interference on external genitalia programming. Reproductive Toxicology 32:1–14. Takeda T, Fujii M, Taura J, Ishii Y, Yamada H. 2012. Dioxin silences gonadotropin expression in perinatal pups by inducing histone deacetylases: A new insight into the mechanism for the im- printing of sexual immaturity by dioxin. Journal of Biological Chemistry 287(22):18440–18450. Tango T, Fujita T, Tanihata T, Minowa M, Doi Y, Kato N, Kunikane S, Uchiyama I, Tanaka M, U ­ ehata T. 2004. Risk of adverse reproductive outcomes associated with proximity to municipal solid waste incinerators with high dioxin emission levels in Japan. Journal of Epidemiology 14(3):83–93. ten Tusscher GW, Stam GA, Koppe JG. 2000. Open chemical combustions resulting in a local in- creased incidence of orofacial clefts. Chemosphere 40(9-11):1263–1270. Townsend JC, Bodner KM, Van Peenen PFD, Olson RD, Cook RR. 1982. Survey of reproductive events of wives of employees exposed to chlorinated dioxins. American Journal of Epidemiol- ogy 115:695–713. Troudi A, Soudani N, Samet AM, Amara IB, Zeghal N. 2011. 2,4-dichlorophenoxyacetic acid effects on nephrotoxicity in rats during late pregnancy and early postnatal periods. Ecotoxicology and Environmental Safety 74:2316–2323.

OCR for page 726
772 VETERANS AND AGENT ORANGE: UPDATE 2012 van den Berg M, Birnbaum LS, Denison M, De Vito M, Farland W, Feeley M, Fiedler H, ­ akansson H H, Hanberg A, Haws L, Rose M, Safe S, Schrenk D, Tohyama C, Tritscher A, Tuomisto J, Tysklind M, Walker N, Peterson RE, 2006. The 2005 World Health Organization reevaluation of human and mammalian toxic equivalency factors for dioxins and dioxin-like compounds. Toxicological Sciences. 93(2):223–241. van Wijngaarden E, Stewart PA, Olshan AF, Savitz DA, Bunin GR. 2003. Parental occupational exposure to pesticides and childhood brain cancer. American Journal of Epidemiology 157(11):989–997. Virtanen HE, Koskenniemi JJ, Sundqvist E, Main KM, Kiviranta H, Tuomisto JT, Tuomisto J, V ­ iluksela M, Vartiainen T, Skakkebaek NE, Toppari J. 2012. Associations between congenital cryptorchidism in newborn boys and levels of dioxins and PCBs in placenta. International Journal of Andrology 35(3):283–293. Wang SL, Lin CY, Guo YL, Lin LY, Chou WL, Chang LW. 2004. Infant exposure to polychlorinated dibenzo-p-dioxins, dibenzofurans and biphenyls (PCDD/Fs, PCBs)—correlation between pre- natal and postnatal exposure. Chemosphere 54:1459–1473. Wang SL, Su PH, Jong SB, Guo YL, Chou WL, Päpke O. 2005. In utero exposure to dioxins and polychlorinated biphenyls and its relations to thyroid function and growth hormone in newborns. Environmental Health Perspectives 113:1645–1650. Weisglas-Kuperus N, Patandin S, Berbers GAM, Sas TCJ, Mulder PGH, Sauer PJJ, Hooijkaas H. 2000. Immunologic effects of background exposure to polychlorinated biphenyls and dioxins in Dutch preschool children. Environmental Health Perspectives 108(12):1203–1207. Wen WQ, Shu XO, Steinbuch M, Severson RK, Reaman GH, Buckley JD, Robison LL. 2000. Paternal military service and risk for childhood leukemia in offspring. American Journal of Epidemiol- ogy 151(3):231–240. Weselak M, Arbuckle TE, Foster W. 2007. Pesticide exposures and developmental outcomes: The epidemiological evidence. Journal of Toxicology and Environmental Health (Part B) 10:41–80. Weselak M, Arbuckle TE, Wigle DT, Walker MC, Krewski D. 2008. Pre-and post-conception pesti- cide exposure and the risk of birth defects in an Ontario farm population. Reproductive Toxicol- ogy 25(4):472–480. WHO (World Health Organization). 1999. Laboratory Manual for the Examination of Human Semen and Sperm-Cervical Mucus Interaction. 4th ed. Cambridge, UK: Cambridge University Press. Wigle DT, Arbuckle TE, Walker M, Wade MG, Liu S, Krewski D. 2007. Environmental hazards: Evidence for effects on child health. Journal of Toxicology and Environmental Health (Part B) 10:3–39. Wigle DT, Arbuckle TE, Turner MC, Berube A, Yang Q, Liu S, Krewski D. 2008. Epidemiologic evidence of relationships between reproductive and child health outcomes and environmental chemical contaminants. Journal of Toxicology and Environmental Health (Part B) 11:373–517. Wolfe WH, Michalek JE, Miner JC, Rahe AJ, Moore CA, Needham LL, Patterson DG Jr. 1995. Paternal serum dioxin and reproductive outcomes among veterans of Operation Ranch Hand. Epidemiology 6:17–22. Wu Q, Ohsako S, Ishimura R, Suzuki JS, Tohyama C. 2004. Exposure of mouse preimplantation em- bryos to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) alters the methylation status of imprinted genes H19 and Igf2. Biological Reproduction 70(6):1790–1797. Yoshioka W, Aida-Yasuoka K, Fujisawa N, Kawaguchi T, Ohsako S, Hara S, Uematsu S, Akira S, Tohyama C. 2012. Critical role of microsomal prostaglandin E synthase-1 in the hydronephro- sis caused by lactational exposure to dioxin in mice. Toxicological Sciences 127(2):547–554. Yuan X, Liu L, Pu Y, Zhang X, He X, Fu Y. 2012. 2,3,7,8-Tetrachlorodibenzo-p-dioxin induces a proteomic pattern that defines cleft palate formation in mice. Food and Chemical Toxicology 50:2270–2274.