Below is the uncorrected machine-read text of this chapter, intended to provide our own search engines and external engines with highly rich, chapter-representative searchable text of each book. Because it is UNCORRECTED material, please consider the following text as a useful but insufficient proxy for the authoritative book pages.
7 Reproductive Effects and Impacts on Future Generations This chapter summarizes the scientific literature published since Veterans and Agent Orange: Update 2006, hereafter referred to as Update 2006 (IOM, 2007), on the association between exposure to herbicides and adverse reproduc- tive or developmental effects. (Analogous shortened names are used to refer to the updates for 1996, 1998, 2000, 2002, and 2004 [IOM, 1996, 1999, 2001, 2003, 2005].) The categories of association and the approach to categorizing the health outcomes are discussed in Chapters 1 and 2. The literature considered in this chapter includes studies of a broad spectrum of reproductive effects in Vietnam veterans or other populations occupationally or environmentally exposed to the herbicides sprayed in Vietnam or to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Because some polychlorinated biphenyls (PCBs) and polychlorodibenzofurans (PCDFs) have dioxin-like biologic activity, studies of populations exposed to PCBs or PCDFs were reviewed if their results were presented in terms of toxicity equivalence quotients (TEQs). As in previous updates, the adverse outcomes evaluated include impaired fertility (in which endometriosis or declines in sperm quality may be involved), increased fetal loss (spontaneous abortion and stillbirth) or neonatal and infant mortality, and other adverse birth outcomes (including low birth weight, preterm birth, and birth defects). In addition to the more delayed problem of childhood cancer in their offspring, this update also addresses the concern of Vietnam vet- erans that their military exposures may contribute to other problems that their children experience later in life or are manifested in later generations. To reduce repetition throughout the report, Chapter 5 presented design in- formation on new studies that report findings on multiple health outcomes. To provide context for publications that present new results on study populations 435
436 VETERANS AND AGENT ORANGE: UPDATE 2008 that were addressed in publications reviewed in earlier updates, Chapter 5 also discussed the overall characteristics of those populations with details about de- sign and analysis relevant to individual papers. For new studies that report only reproductive health outcomes and that are not revisiting previously studied popu- lations, design information is summarized in this chapter with results. This chapterâs primary emphasis is on the potential adverse reproductive effects of herbicide exposure of men because the vast majority of Vietnam vet- erans are men. However, about 8,000 women served in Vietnam (H. Kang, US Department of Veterans Affairs, personal communication, December 14, 2000), so findings relevant to female reproductive health are also included. Whenever the information was available, an attempt was made to evaluate the effects of maternal and paternal exposure separately. Exposure scenarios in human popula- tions and experimental animals studied differ in their applicability to our popula- tion of concern according to whether the exposed parent was a male or female veteran. In addition, for published epidemiologic or experimental results to be fully relevant to evaluation of the plausibility of reproductive effects in Vietnam veterans, female as well as male, the timing of exposure needs to correspond to the veteransâ experience (that is, occur only prior to 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. BIOLOGIC PLAUSIBILITY OF REPRODUCTIVE EFFECTS This chapter opens with a general discussion of factors that influence the plausibility that TCDD and the four herbicides used in Vietnam could produce adverse reproductive effects. There have been very few reproductive 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 pre- ponderance of the following discussion concerns TCDD. Because dioxin is stored in fat tissue and has a very long biologic half-life, internal exposure at generally constant concentrations may continue after epi- sodic, high-level exposure to external sources has ceased. If a person had high ex- posure, there may still be high amounts of dioxin stored in fat tissue, which may be mobilized, particularly at times of weight loss. That would not be expected to be the case for nonlipophilic chemicals, such as cacodylic acid. The paternal contribution to a pregnancy is limited to the contents of the sperm that fertilizes an egg, any damage would be conveyed as DNA mutations or epigenetic effects (that is, heritable changes in genome function that occur without a change in primary DNA sequences). Dioxins have not been shown to alter DNA sequences (they do not produce mutations), so the potential effects in offspring are limited to epigenetic effects. Two possible mechanisms could theoretically produce affected children: if sperm stem cells are altered by expo-
REPRODUCTIVE EFFECTS AND IMPACTS ON FUTURE GENERATIONS 437 sure, they could continue to produce altered sperm; and mobilization of dioxin from storage in adipose tissue (for example, due to weight loss) could continue to damage a manâs developing sperm, and thus interfere with conception and conceptuses. In any case, any exposure of the father that could affect his children must occur before their conception. Although ova, the maternal contribution to a conceptus, do not undergo the repeated 90-day cycles of spermatogenesis, they might be damaged by modes of exposure analogous to those affecting male gametes. In addition, at critical peri- ods of gestation and even postnatally through breast milk, the female can mediate exposure to the offspring as it develops. Such exposure can interfere with cell replication, differentiation, and migration; with formation of tissues, organs, and systems; and with structure. Dioxin in her bloodstream, whether from external sources or released from fat stores, can cross the placenta and expose the develop- ing embryo and fetus. Mobilization of dioxin during pregnancy or lactation may be increased because the body is drawing on fat stores to supply nutrients to the developing fetus or nursing infant. Breast milk has a high fat content, and the concentration of dioxin in breast milk is about 100 times that in a motherâs blood. In animal studies, TCDD crosses the placenta and is transferred via breast milk. In humans, TCDD has been measured in circulating 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 that in an infant not breastfed (Lorber and Phillips, 2002). Thus, the exposure of human infants to TCDD in utero and via lactation has been demonstrated. Toxicologists often distinguish between reproductive effects, which concern the reproductive process itself, and developmental effects, which involve differ- entiation of fetal tissues and maturation of the offspring. A connection between TCDD exposure and human reproductive and developmental effects is, in general, biologically plausible. However, more definitive conclusions about the potential for such TCDD toxicity in humans are complicated by differences in sensitivity and susceptibility among individual animals, strains, and species; by the lack of strong evidence of organ-specific effects among species; by differences in route, dose, duration, and timing of exposure; and by substantial differences in the toxicokinetics of TCDD between laboratory animals and humans. Experiments with 2,4-dichlorophenoxyacetic acid (2,4-D) and 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) indicate that they have subcellular effects that could constitute a biologically plausible mechanism for reproductive and developmental effects. Evidence from animals, however, indicates that they do not have reproductive effects and that they have developmental effects only at very high doses. There is insufficient information on picloram and cacodylic acid to assess the biologic plausibility of those compoundsâ reproductive or developmental effects. The biologic plausibility portions of sections on the specific outcomes con- sidered in this chapter present more detailed toxicologic findings that are of particular relevance to the outcomes discussed.
438 VETERANS AND AGENT ORANGE: UPDATE 2008 ENDOMETRIOSIS Endometriosis (International Classification of Diseases, 9th revision [ICD-9], code 617) affects 5.5 million women in the United States and Canada at any given time (NICHD, 2007). The endometrium is the tissue that lines the inside of the uterus and is built up and shed each month during menstruation. In endometrio- sis, endometrial cells are found outside the uterusâusually in other parts of the reproductive system, in the abdomen, or on surfaces near the reproductive organs. That misplaced tissue develops into growths or lesions that continue to respond to hormonal changes in the body and break down and bleed each month in concert with the menstrual cycle. Unlike blood released during normal shedding of the endometrium lining the uterus, blood released in endometriosis has no way to leave the body, and the results are inflammation, internal bleeding, and degenera- tion of blood and tissue that can cause scarring, pain, infertility, adhesions, and intestinal problems. There are several theories of the etiology of endometriosis, including a ge- netic contribution, but the cause remains unknown. Estrogen dependence and im- mune modulation are established features of endometriosis but do not adequately explain the cause of this disorder. It has been proposed that endometrium is distributed through the body via blood or the lymphatic system; that menstrual tissue backs up into the fallopian tubes, implants in the abdomen, and grows; and that all women experience some form of tissue backup during menstruation but only those with immune-system or hormonal problems experience the tissue growth associated with endometriosis. Despite numerous symptoms that can indicate endometriosis, diagnosis is possible only through laparoscopy or a more invasive surgical technique. Several treatments for endometriosis are available, but there is no cure. Conclusions from VAO and Updates Endometriosis was first reviewed in this series of reports in Update 2002, which identified two relevant environmental studies, and Update 2004 examined three environmental studies. Two additional environmental studies considered in Update 2006 did not change the conclusion that the evidence was inadequate or insufficient to support an association with herbicide exposure. Table 7-1 provides a summary of relevant studies that have been reviewed. Update of the Epidemiologic Literature No new Vietnam-veteran or occupational studies addressing endometriosis have been published since Update 2006.
REPRODUCTIVE EFFECTS AND IMPACTS ON FUTURE GENERATIONS 439 TABLE 7-1â Selected Epidemiologic StudiesâEndometriosis Reference Study Population Study Results ENVIRONMENTAL New Studies Heilier Serum DLC and aromatase No association between TEQs of DLCs in serum et al., activity in endometriotic tissue and aromatase activity by regression analyses. 2006 from 47 patients in Belgium p-values = 0.37â0.90 for different endometriosis subgroups. Heilier 88 matched triads (264 total); Results for pelvic endometriosis vs controls et al., patients with deep endometriotic Dietary fat: OR = 1.0 (95% CI 1.0â1.0) 2007 nodules, pelvic endometriosis, BMI: OR = 1.0 (95% CI 0.9â1.0) controls matched for age, Occupation: OR = 0.5 (95% CI 0.2â1.1) gynecologic practice in Belgium; Traffic: OR = 1.0 (95% CI 0.3â2.8) routes of exposure to DLCs Incinerator: OR = 1.0 (95% CI 1.0â1.1) examined Tsuchiya 138 infertility patients in Japan; Results for advanced endometriosis et al., laproscopically confirmed caseâ Total TEQ: OR = 0.5 (95% CI 0.2â1.7) 2007 control status, serum dioxin, PCB Genotype-specific: ORs = 0.3â0.6 TEQ; P450 genetic polymorphism significant interaction between genotype, No dioxin TEQ Studies Reviewed in Update 2006 Heilier Endometriosis in Belgian women 50 exposed cases, risk of increase of 10 pg/p lipid et al., with overnight fasting serum of TEQ compounds: OR = 2.6 (95% CI 1.3â5.3) 2005 levels of PCDD, PCDF, PCB Porpora Caseâcontrol study of Italian Mean total PCBs (ng/g) et al., women with endometriosis, Cases, 410 ng/g 2006 measured serum PCBs Control, 250 ng/g PCB congeners: OR = 4.0 (95% CI 1.3â13) All Studies Review in Update 2004 De Felip Pilot study of Italian, Belgian Mean concentration of TCDD (ppt of lipid) et al., women of reproductive age; Italy: 2004 compared concentrations of Controls (10 pooled samples), 1.6 TCDD, total TEQ in pooled Cases (two sets of six pooled samples), 2.1, 1.3 blood samples from women who Belgium: had diagnosis endometriosis with Controls (seven pooled samples), 2.5 controls Cases (Set I, five pooled samples; Set II, six pooled samples), 2.3, 2.3 Mean concentration of TEQ (ppt of lipid) Italy: Controls (10 pooled samples), 8.9 Â± 1.3 (99% CI 7.2â11) Cases (two sets of six pooled samples), 10.7 Â± 1.6; 10.1 Â± 1.5 Belgium: Controls (seven pooled samples), 24.7 Â± 3.7 (99% CI 20â29) Cases (Set I, five pooled samples; Set II, six pooled samples), 18.1 Â± 2.7; 27.1 Â± 4 .0 continued
440 VETERANS AND AGENT ORANGE: UPDATE 2008 TABLE 7-1â Continued Reference Study Population Study Results Fierens Belgian women with Mean concentration of TEQ a (ppt of lipid) et al., environmental exposure to Cases (n = 10), 26.2 (95% CI 18.2â37.7) 2003 PCDDs, PCDFs; compared Controls (n = 132), 25.6 (95% CI 24.3â28.9) analyte concentrations in cases vs No significant difference controls Eskenazi Residents of Seveso Zones A Serum TCDD (ppt) et al., and B up to 30 years old in 20.1â100 ppt (n = 8), OR = 1.2 2002 1976; compared incidence of (90% CI 0.3â4.5) endometriosis across serum > 100 ppt (n = 9), OR = 2.1 (90% CI 0.5â8.0) TCDD concentrations Studies Reviewed in Update 2002 Pauwels Patients undergoing infertility Six exposed cases: OR = 4.6 (95% CI 0.5â43.6) et al., treatment in Belgium; compared 2001 number of women with, without endometriosis who had serum dioxin levels up to 100 pg TEQ/g of serum lipid Mayani Residents of Jerusalem being Eight exposed cases: OR = 7.6 et al., evaluated for infertility; (95% CI 0.9â169.7) 1997 compared number of women with high TCDD who had (n = 44), did not have (n = 35) diagnosis of endometriosis ABBREVIATIONS: BMI, body mass index; CI, confidence interval; DLC, dioxin-like compound; OR, odds ratio; PCB, polychlorinated biphenyl; PCDD, polychlorinated dibenzodioxin; PCDF, polychlorinated dibenzofuran; TCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxin; TEQ, toxicity equivalent quotient. aTEQs calculated using the 1998 World Health Organization dioxin toxic equivalency factor (TEF) method (Van den Berg et al., 1998). Environmental Studies Three new studies concerning exposure to the compounds of interest and endometriosis have been conducted since the last update. The first two were conducted by the same research group that reported an increased risk of endo- metriosis with serum concentrations of dioxin-like compounds (DLCs) (Heilier et al., 2005). In the new studies, they sought to expand their findings by focusing on a possible biologic pathway (aromatase activity) and routes of exposure (such as diet and residential proximity to waste incinerators). However, neither study showed significant associations of endometriosis with the factors studied. The third new study (Tsuchiya et al., 2007), which looked at specific genes in addi- tion to dioxin exposure, did not find an association between dioxin exposure and early stage-endometriosis regardless of genotype. In contrast with some earlier
REPRODUCTIVE EFFECTS AND IMPACTS ON FUTURE GENERATIONS 441 studies, it found dioxin exposure to be associated with a lower risk of advanced endometriosis. This lower risk was particularly strong in women who had a specific genotype. In the study of Heilier et al. (2006), 47 women admitted to a university hospital in Belgium for the treatment of endometriosis agreed to participate in a study aimed at determining whether aromatase activity was associated with the concentration of DLCs in the endometriotic tissue. Aromatase is an enzyme im- portant in the synthesis of estrogen, and drugs that block the synthesis of estrogen lead to a reduction in endometriotic tissue. The authors thought that DLCs might increase the aromatase, which would increase estrogen and lead to increased growth of endometrial tissue. Endometriotic tissue was surgically removed from the women, and DLCs and aromatase activity were measured in the laboratory. They found that the concentration of DLCs was not associated with higher aro- matase activity in endometriotic tissue. Heilier et al. (2007) studied a total of 264 women in the same gynecologic practice, divided equally into three groups matched on age; cases of pelvic endometriosis, cases of deep endometriotic nodules, and controls. Serum TEQ concentrations were available for 58 of these women who had participated in a previous study by the authors (Heilier et al., 2005), which found a risk of endo- metriosis associated with exposure to DLCs. Interviews conducted with patients and controls collected information on diet, occupation, and residential proximity to automobile traffic, waste incinerators, or other pollution sources. In the subset of women whose serum DLCs were measured, they found that those with higher concentrations were more likely to have consumed specific high-fat foods: pig meat, marine fish, and fresh cream. However, neither dietary fat consumption, body mass index (BMI), residential proximity to automobile traffic or waste incinerators, nor specific occupation was associated with pelvic endometriosis or with deep endometriotic nodules. The study used indirect measures of dioxin exposure that are expected to be less precise than measures of serum dioxin, so the results do not necessarily contradict those of their earlier study. The investi- gators were unable to identify any likely source of dioxin exposure that differed between cases and controls. The third new study was conducted by Tsuchiya et al. (2007) in Japan to ex- amine a possible association between genetic susceptibility to effects of exposure to DLCs and endometriosis. They studied a total of 138 women who sought treat- ment for infertility and had undergone laparoscopy. On the basis of laparoscopy, the women were classified as having early-stage endometriosis (stages IâII), ad- vanced endometriosis (stages IIIâIV), or no evidence of endometriosis (controls). They measured serum dioxin and DLCs (TEQ per gram of lipids) and extracted DNA from serum to determine polymorphisms (different genetic versions) of two genes (cytochromes P450 [CYP] 1A1 and 1B1), which regulate the synthesis and metabolism of endogenous and exogenous estrogens. They hypothesized that dif- ferences in genetic makeup might confer differences in susceptibility to effects of
442 VETERANS AND AGENT ORANGE: UPDATE 2008 DLCs and might explain why studies of endometriosis and dioxins show incon- sistent results. Overall, serum-dioxin concentrations did not differ significantly between cases with early or advanced endometriosis and controls (adjusted for age). Women who had advanced endometriosis were less likely to have high serum dioxins than controls (OR = 0.46, 95% CI 0.20â1.06), but the difference was of borderline statistical significance; the result was virtually unchanged by considering the total concentration of PCBs and dioxins. When genotype was considered, the authors found no significant interaction between genotype and serum dioxin in women who had early endometriosis. However, there was some evidence of interaction between CYP genes and dioxin exposure with respect to the risk of advanced endometriosis. There was a reduced risk of advanced endo- metriosis after high dioxin exposure in women who had the less common allele for CYP1A1, but the number of women in this group was very small. In summary, the study found some evidence of a geneâenvironment interaction related to the occurrence of endometriosis, but women who had higher concentrations of dioxin were found to be at lower risk for advanced endometriosis. Biologic Plausibility Laboratory studies that used animal models and examined gene-expression changes associated with human endometriosis and TCDD exposure provide evi- dence to support the biologic plausibility of a link between TCDD exposure and endometriosis. The first suggestion that TCDD exposure may be linked to endo- metriosis came as a secondary finding from a study that exposed female rhesus monkeys (Macaca mulatta) chronically to low concentrations of dietary TCDD for 4 years (Rier et al., 1993). Ten years after the exposure ended, the investiga- tors documented an increased incidence of endometriosis in the monkeys that correlated with the dioxin exposure concentration. The small sample prevented a definitive conclusion that TCDD was a causal agent in the development of the endometriosis, but it led to numerous studies of the ability of TCDD to promote the growth of pre-existing endometriotic lesions. When fragments of uterine tissue were implanted in the peritoneal cavity to mimic eutopic endometrial lesions, TCDD exposure was shown to promote the survival and growth of the lesions in monkeys and in rodents (Cummings et al., 1996; Johnson et al., 1997; Yang et al., 2000). In mice, direct treatment of endo- metrial tissue with TCDD before placement into the peritoneal cavity resulted in increased size and number of endometrial lesions (Bruner-Tran et al., 1999). A number of proposed mechanisms by which TCDD may promote endome- trial lesions provide additional biologic plausibility of the link between TCDD and endometriosis. Human endometrial tissue expresses the aryl hydrocarbon receptor (AHR) and its dimerization partner, the aryl hydrocarbon nuclear trans- locator (ARNT) (Khorram et al., 2002), and three AHR target genes: CYP1A1,
REPRODUCTIVE EFFECTS AND IMPACTS ON FUTURE GENERATIONS 443 1A2, and 1B1 (Bulun et al., 2000). That suggests that endometrial tissue is respon- sive to TCDD. Furthermore, TCDD significantly decreases the ratio of progester- one receptor B to progesterone receptor A in normal human endometrial stromal cells and blocks the ability of progesterone to suppress matrix metalloproteinase (MMP) expression; these actions may promote endometrial-tissue invasion. Both the reduced ratio and the resistance to progesterone-mediated MMP suppression are observed in endometrial tissue from women who have endometriosis (Igarashi et al., 2005). Progesterone prevents endometrial breakdown before menstruation by down-regulating expression of endometrial matrix metalloproteins during the secretory phase of the menstrual cycle. Bruner-Tran et al. (2008) have proposed that environmental toxicants, such as TCDD, that disrupt progesterone action may predispose the endometrium to an inflammatory microenvironment that would promote a process of tissue loss at menstruation. Their hypothesis is supported by their evidence that TCDD inhibits expression of progesterone receptor and transforming growth factor Î²2 in the endometrium and possibly expression of other immune modulators regulated by progesterone. TCDD induces changes in gene expression that mirror those observed in endometrial lesions. For example, TCDD can induce expression of histamine- releasing factor, which is increased in endometrial lesions and accelerates their growth (Oikawa et al., 2002, 2003). Similarly, TCDD stimulates expression of RANTES (regulated on activation, normal T cellâexpressed, and secreted) in endometrial stromal cells, and RANTES concentration and bioactivity are in- creased in women who have endometriosis (Zhao et al., 2002). The two CC-motif chemokines (chemotactic cytokines), RANTES and macrophage-inflammatory protein (MIP)-1Î±, have been identified as potential contributors to the pathogen- esis and progression of endometriosis. To probe the effect of dioxin exposure and estrogen on expression of those chemokines in endometriosis-associated cells and to explore the pathogenesis of endometriosis, endometrial stromal cells were exposed to a combination of 17Î²-estradiol and TCDD. The combined treatment increased the secretion of RANTES and MIP-1Î±, promoted the invasiveness of endometrial stromal cells, and increased the expression of matrix metallopro- teins MMP-2 and MMP-9 in endometrial stromal cells, indicating that combined TCDD and estradiol may facilitate the onset of endometriosis and contribute to its development by increasing the invasion of endometrial stromal cells medi- ated by CC-motif chemokines (Yu et al., 2008). Those data are consistent with the evidence of an interaction between the AHR and the estrogen receptor that induces estrogen-mediated proliferative effects in the mouse uterus (Ohtake et al., 2008). Differences between the mouse uterus and the human endometrium pre- vent absolute extrapolation, but the data suggest that dioxins may induce changes in endometrial physiology. In summary, it may be expected that TCDD exposure would create an inflammatory endometrial microenvironment that could disrupt endometrial function and cause disease.
444 VETERANS AND AGENT ORANGE: UPDATE 2008 Although those studies do not establish the degree to which TCDD may cause or promote endometriosis, they do provide evidence that supports the bio- logic plausibility of a link between TCDD exposure and endometriosis. Synthesis The three new studies described above were designed to follow on previous studies showing an association between DLCs and endometriosis. Two of the studies (Heilier et al., 2006, 2007) were conducted by the same research group that had found a significant association between blood concentrations of DLCs and risk of endometriosis. They sought to shed light on possible biologic path- ways and routes of exposure that might expand on the previous findings. How- ever, neither the evaluation of an enzyme important in the synthesis of estrogen nor examination of routes of exposure to dioxin yielded evidence of association with endometriosis. The third new study (Tsuchiya et al., 2007) found a decreased risk of endometriosis in women with higher dioxin concentrations. Overall, the studies linking dioxin exposure with endometriosis are few in number and inconsistent. The association in animal studies is biologically plau- sible, but it is possible that human exposures are too low to show an association consistently. Conclusion On the basis of the evidence reviewed here and in the previous VAO reports, the committee concludes that there is inadequate or insufficient evidence to sup- port an association between exposure to the chemicals of interest and human endometriosis. FERTILITY Male reproductive function is under the control of several components whose proper coordination is important for normal fertility. Several of the components and some health outcomes related to male fertility, including reproductive hor- mones and sperm characteristics, can be studied as indicators of fertility. The reproductive neuroendocrine axis involves the central nervous system, the an- terior pituitary gland, and the testis. The hypothalamus integrates neural inputs from the central and peripheral nervous systems and regulates the gonadotropins luteinizing hormone (LH) and follicle-stimulating hormone (FSH). Both are se- creted into the circulation in episodic bursts by the anterior pituitary gland and are necessary for normal spermatogenesis. In the testis, LH interacts with receptors on Leydig cells, where it stimulates increased testosterone synthesis. FSH and the testosterone from the Leydig cells interact with the Sertoli cells in the semi- niferous tubule epithelium to regulate spermatogenesis. More detailed reviews
REPRODUCTIVE EFFECTS AND IMPACTS ON FUTURE GENERATIONS 445 of the male reproductive hormones can be found elsewhere (Knobil et al., 1994; Yen and Jaffe, 1991). Several agents, such as lead and dibromochloropropane, affect the neuroendocrine system and spermatogenesis (for reviews, see Bonde and Giwercman, 1995; Tas et al., 1996). Studies of the relationship between chemicals and fertility are less common in women than in men. Some chemicals may disrupt the female hormonal bal- ance necessary for proper functioning. Normal menstrual-cycle functioning is also important in the risk of hormonally related diseases, such as osteopenia, breast cancer, and cardiovascular disease. Chemicals can have multiple effects on the female system, including modulation of hormone concentrations result- ing in menstrual-cycle or ovarian-cycle irregularities, changes in menarche and menopause, and impairment of fertility (Bretveld et al., 2006a,b). In this section, we also discuss studies that have focused on menstrual-cycle characteristics and age at menarche or menopause. An affect on age at menarche would be of concern among the daughters of Vietnam veterans rather than among female veterans themselves, but the occurrence of this effect in other populations would demonstrate the ability of the chemicals in question to perturb functioning of the female reproductive system. Conclusions from VAO and Updates The committee responsible for the original VAO report (IOM, 1994) con- cluded that there was inadequate or insufficient evidence of an association be- tween exposure to 2,4-D, 2,4,5-T, TCDD, picloram, or cacodylic acid and altered sperm characteristics or infertility. Overall, additional information available to the committees responsible for Update 1996, Update 1998, Update 2000, Update 2002, Update 2004, and Update 2006 did not change that finding. Reviews of the relevant studies are presented in the earlier reports. Tables 7-2 and 7-3 summarize the studies related to male and female fertility, respectively. Update of the Epidemiologic Literature Male Fertility Vietnam-Veteran Studiesâ One new Vietnam-veteran study has been published since Update 2006. Gupta et al. (2006) compared serum testosterone concentra- tions measured in 1987 with TCDD concentrations measured at the same time in veterans in the Air Force Health Study. A total of 971 Ranch Hand veterans and 1,266 comparison veterans with serum TCDD and serum testosterone measure- ments were included in the analyses. After adjustment for age and BMI in 1987 and for a percentage change in BMI from the end of their Southeast Asia tour to 1987, higher serum TCDD was significantly associated with lower testoster- one concentrations in both Ranch Hands (slope for ln[TCDD] = -0.02, 95% CI
446 VETERANS AND AGENT ORANGE: UPDATE 2008 TABLE 7-2â Selected Epidemiologic StudiesâMale Fertility (Altered Hormone Concentrations, Decreased Sperm Counts or Quality, Subfertility, or Infertility) Exposed Measure of Risk Reference Study Population Casesa (95% CI)a VIETNAM VETERANS New Studies Gupta AFHS (964 Ranch Hands, 1,259 comparison) Coefficient (p-value) et al., for ln(Testosterone) 2006 vs ln(TCDD) in 1987 Comparison TCDD quartile I (mean, 2.14 ppt) nr 0 (referent) Comparison TCDD quartile II (mean, 3.54 ppt) nr -0.063 (0.004) Ranch Hand TCDD quartile I (mean, 4.14 ppt) nr 0.002 (0.94) Comparison TCDD quartile III (mean, 4.74 ppt) nr -0.048 (0.03) Comparison TCDD quartile IV (mean, 7.87 ppt) nr -0.079 (< 0.001) Ranch Hand TCDD quartile II (mean, 8.95 ppt) nr -0.052 (0.03) Ranch Hand TCDD quartile III (mean, 18.40 ppt) nr -0.029 (0.22) Ranch Hand TCDD quartile IV (mean, 76.16 ppt) nr -0.056 (0.02) Studies Reviewed in Update 1996 Henriksen Effects on specific hormone concentrations or et al., sperm count in Ranch Hands 1996 Low testosterone High dioxin (1992) 18 1.6 (0.9â2.7) High dioxin (1987) 3 0.7 (0.2â2.3) Low dioxin (1992) 10 0.9 (0.5â1.8) Low dioxin (1987) 10 2.3 (1.1â4.9) Background (1992) 9 0.5 (0.3â1.1) High FSH High dioxin (1992) 8 1.0 (0.5â2.1) Low dioxin (1992) 12 1.6 (0.8â3.0) Background (1992) 16 1.3 (0.7â2.4) High LH High dioxin (1992) 5 0.8 (0.3â1.9) Low dioxin (1992) 5 0.8 (0.5â3.3) Background (1992) 8 0.8 (0.4â1.8) Low sperm count High dioxin 49 0.9 (0.7â1.2) Low dioxin 43 0.8 (0.6â1.0) Background 66 0.9 (0.7â1.2) Studies Reviewed in VAO CDC, Vietnam Experience Study 1989 Lower sperm concentration 42 2.3 (1.2â4.3) Proportion of abnormal sperm 51 1.6 (0.9â2.8) Reduced sperm motility 83 1.2 (0.8â1.8) Stellman American Legionnaires who served in SEA et al., Difficulty in having children 349 1.3 (p < 0.01) 1988
REPRODUCTIVE EFFECTS AND IMPACTS ON FUTURE GENERATIONS 447 TABLE 7-2â Continued Exposed Measure of Risk Reference Study Population Casesa (95% CI)a OCCUPATIONAL Newly considered study Egeland Male chemical workers exposed to dioxin vs Risk of extreme et al., neighborhood controls in New Jersey, Missouri hormone 1994 measured in 1987 concentration Testosterone (< 10.4 nmol/L) Referents (TCDD < 20 ppt) 11 1.0 Workers 25 2.1 (1.0â4.6) Quartile I (TCDD < 20 ppt) 2 0.9 (0.2â4.5) Quartile II (TCDD 20â75 ppt) 7 3.9 (1.3â11.3) Quartile III (TCDD 76â240 ppt) 6 2.7 (0.9â8.2) Quartile IV (TCDD 241â3,400 ppt) 10 2.1 (0.8â5.8) FSH (> 31 IU/L) 20 1.5 (0.7â3.3) LH (> 28 IU/L) 23 1.6 (0.8â3.3) Studies Reviewed in Update 2006 Oh et al., Male fertility, dioxin exposure with air 2005 monitoring 31 1.4 (nr) Studies Reviewed in Update 2000 Larsen Danish farmers who used any potentially et al., spermatotoxic pesticides, including 2,4-D 1998 Farmers using pesticides vs organic farmers 523 1.0 (0.8â1.4)b Used three or more pesticides nr 0.9 (0.7â1.2)b Used manual sprayer for pesticides nr 0.8 (0.6â1.1)b Studies Reviewed in Update 1998 Heacock Workers at sawmills using chlorophenates et al., Standardized fertility ratio 18,016 0.7 (0.7â0.8)c 1998 (births) MantelâHaenszel rate-ratio estimator 18,016 0.9 (0.8Â-0.9)c (births) Cumulative exposure (hours) 120â1,999 7,139 0.8 (0.8â0.9)c 2,000â3,999 4,582 0.9 (0.8â1.0)c 4,000â9,999 4,145 1.0 (0.9â1.1)c â¥ 10,000 1,300 1.1 (1.0â1.2)c Lerda and Argentinean farmers exposed to 2,4-D 32 Rizzi, Sperm count (millions/mL) exposed: 49.0 vs control: 101.6 1991 Motility (%) exposed: 24.8 vs control: 70.4 Sperm death (%) exposed: 82.9 vs control: 37.1d Anomalies (%) exposed: 72.9 vs control: 33.4 (p < 0.01 overall) ENVIRONMENTAL New Studies Cok et al, Caseâcontrol study of infertile men in Ankara, 22 fertile 9.4 TEQ pg/g lipid 2008 Turkey; adipose-tissue samples assayed for 23 infertile 12.5 TEQ pg/g lipid dioxins, furans, dl PCBs (p = 0.065) Mocarelli Men exposed in Seveso, Zone A vs age-matched et al., men residing outside the contamination zone, 2008 measured semen characteristics, estradiol, FSH, testosterone, LH, inhibin B Authorsâ evaluation Age at 1976 exposure: (data not shown) Infant/prepuberty (1â9 year), n = 71, 176 Sensitive continued
448 VETERANS AND AGENT ORANGE: UPDATE 2008 TABLE 7-2â Continued Exposed Measure of Risk Reference Study Population Casesa (95% CI)a Puberty (10â17 year), n = 44, 136 Intermediate response Adult (18â26 year), n = 20, 60 No associations Polsky Caseâcontrol study of erectile dysfunction in Highest vs lowest et al., urology patients patients in Ontario, Canada PCB groups 2007 PCB-118 (TEF = 0.0001) 1.0 (0.5â2.1) PCB-156 (TEF = 0.0005) 0.9 (0.5â1.6) PCB-170 0.6 (0.3â1.2) PCB-180 0.7 (0.4â1.4) Toft Men in general population of Poland, Greenland, et al., Ukraine, Sweden; AHR binding measured with 2007 CALUX assay Measurements of semen quality (concentration, No consistent motility, percentage normal) associations Dhooge Men in general population of Belgium; et al., Association with 2-fold increase in 2006 CALUX-TEQ Change (p-value) Sperm concentration 25.2% (p = 0.07) Semen volume â16.0% (p = 0.03) Total testosterone â7.1% (p = 0.04) Free testosterone â6.8% (p = 0.04) Studies Reviewed in Update 2006 Swan Men in Missouri, US with or without low sperm et al., quality 2003 Increased urinary metabolite marker for 2,4-D 5 0.8 (0.2â3.0) Studies Reviewed in Update 2002 Staessen Adolescents in communities close to industrial et al., sources of heavy metals, PCBs, VOCs, and 2001 PAHsâdelays in sexual maturity In Hoboken, Belgium 8 4.0 (nr) In Wilrik, Belgium 15 1.7 (nr) ABBREVIATIONS: 2,4Â-D, 2,4-dichlorophenoxyacetic acid; AFHS, Air Force Health Study; AHR, aryl hydrocarbon receptor; CALUX, assay for determination of dioxin-like activity; CI, confidence interval; dl, dioxin-like; FSH, follicle-stimulating hormone; IU, international unit; LH, luteinizing hormone; nr, not reported; PAH, polycyclic aromatic hydrocarbon; PCB, polychlorinated biphenyl; SEA, Southeast Asia; TCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxin; TEF, toxicity equivalency factor; TEQ, toxicity equivalent quotient; VOC, volatile organic compounds. aGiven when available; results other than estimated risk explained individually. bFor this study, relative risk has been replaced with fecundability ratio, for which value less than 1.0 indicates adverse effect. cFor this study, relative risk has been replaced with standardized fertility ratio, for which value less than 1.0 indicates adverse effect. dTable 1 in reference reverses these figuresâcontrol, 82.9%; exposed, 37.1%âbut text (âThe percent- ages of asthenospermia, mobility, necrosperma and teratospermia were greater in the exposed group than in controls. . .â) suggests that this is a typographical error.
REPRODUCTIVE EFFECTS AND IMPACTS ON FUTURE GENERATIONS 449 TABLE 7-3â Selected Epidemiologic StudiesâFemale Fertility (Altered Hormone Concentrations, Subfertility, or Infertility) Exposed Estimated Relative Risk Reference Study Population Cases (95% CI)a OCCUPATIONAL Studies Reviewed in Update 2006 Farr Age of menopause women who self-reported et al., pesticide exposure 2006 8,038 0.9 (0.8â1.0) Farr Menstrual-cycle characteristics of et al., premenopausal women in AHS 21â40 years old 1,754 2004 Short menstrual cycle 0.8 (0.6â1.0) Long menstrual cycle 1.4 (0.9â2.1) Irregular 0.6 (0.4â0.8) Missed period 1.6 (1.3â2.0) Intermenstrual bleeding 1.1 (0.9â1.4) ENVIRONMENTAL New Studies Chao Pregnant women in Taiwan; measured placental Regression adjusted for et al., dioxin TEQ, PCB TEQ maternal age, BMI, parity 2007 Older of âregular menstrual cycleâ Dioxin TEQ p = 0.032 PCB TEQ p = 0.077 Longer âlongest menstrual cycleâ Dioxin TEQ p = 0.269 PCB TEQ p = 0.006 Eskenazi Seveso Womenâs Health Studyâfibroids et al., among women from Zones A & B newborn to 2007 40 yr in 1976 log10 TCDD (ppt) Age-adjusted hazard ratio â¤ 20.0 1.0 20.1â75.0 0.6 (0.4â0.8) > 75.0 0.6 (0.4â0.9) Warner SWHSâovarian function in women in Zones et al., A and B, newborn to 40 years old in 1976 2007 Ovarian follicles in follicular phase (age- adjusted OR) 1.0 (0.4â2.2) Ovulation (age-adjusted OR) in luteal phase 1.0 (0.5â1.9) in midluteal phase 1.0 (0.4â2.7) Estradiol slopes with log10 TCDD in luteal phase â1.8 (â10.4 to 6.8) in midluteal phase â3.1 (â14.1 to 7.8) Progesterone in luteal phase â0.7 (â2.4 to 1.0) in midluteal phase â0.8 (â3.7 to 2.0) continued
450 VETERANS AND AGENT ORANGE: UPDATE 2008 TABLE 7-3â Continued Exposed Estimated Relative Risk Reference Study Population Cases (95% CI)a Studies Reviewed in Update 2006 Eskanazi Seveso cohort, serum-dioxin concentrations, et al., stage of menopause 616 2005 Premenopause 260 43.6 (0.2â0.9) Natural menopause 169 45.8 (0.3â1.0) Surgical menopause 83 43.4 (0.3â1.0) Impending menopause 13 43.8 (0.2â0.9) Perimenopause 33 36.5 (0.2â0.9) Other 58 39.6 (0.2â0.9) Greenlee Women in Wisconsin, US with or without et al., infertility (maternal exposure) 2003 Mixed or applied herbicides 21 2.3 (0.9â6.1) Used 2,4,5-T 9 cases (2.7%) 9 11 controls (3.4%) Used 2,4-D 4 cases (1.2%) 4 4 controls (1.2%) Warner SWHSâage at menarche et al., 2004 282 1.0 (0.8â1.1) ABBREVIATIONS: 2,4Â-D, 2,4-dichlorophenoxyacetic acid; 2,4,5-T, 2,4,5-trichlorophenoxyacetic acid; AHS, Agricultural Health Study; BMI, body mass index; CI, confidence interval; OR, odds ratio; PCB, polychlorinated biphenyl; SWHS, Seveso Womenâs Health Study; TCDD, 2,3,7,8- tetrachlorodibenzo-p-dioxin; TEQ, toxicity equivalent quotient. aGiven when available; results other than estimated risk explained individually. -0.04 to -0.002) and comparison veterans (slope for ln[TCDD] = -0.05, 95% CI -0.08 to -0.03). Because both TCDD and testosterone measurements were log transformed for analysis, it is difficult to convey the magnitude of the adjusted association; the comparison group had considerably lower TCDD than the Ranch Hands, but the inverse relationship with testosterone appeared at least as strong. The estimated slopes have overlapping confidence intervals, but given that tes- tosterone levels appear more profoundly depressed in the comparison group, it would have been informative to determine the relationship with ln[TCDD] over the full range analyzing the data for both groups together. In the unadjusted quartile distributions, Ranch Hands with the highest exposure had average tes- tosterone of 530 ng/dL, and the least exposed 583 ng/dL. Comparison veterans with the highest exposure had average testosterone of 491 ng/dL, and the least exposed, 606 ng/dL. The lead author (A Gupta, University of Texas Southwestern Medical Center, personal communication on March 29, 2009) confirmed that the single specification of a unit for testosterone concentrationâin Table 2 as ng/mLâwas an error. The adjusted mean testosterone measurements were sig-
REPRODUCTIVE EFFECTS AND IMPACTS ON FUTURE GENERATIONS 451 nificantly lower in the most exposed veterans in both groups, but they were still well within the normal range of 300â1,000 ng/dL for adult men (http://www.nlm. nih.gov/MEDLINEPLUS/ency/article/003707.htm). Environmental Studiesâ To investigate TCDDâs effect on reproductive hor- mones and sperm quality, Mocarelli et al. (2008) compared 135 men exposed to TCDD by the 1976 Seveso accident with 184 healthy men neither exposed to TCDD nor living in the Seveso contamination zones. Each group was di- vided into three categories that reflected age at the time of the Seveso accident: infancyâprepuberty (1â9 years), puberty (10â17 years), and adulthood (18â26 years). The study found that TCDD exposure in infancyâprepuberty was associ- ated with lower sperm concentration and motility in adulthood, lower estradiol, and higher FSH. Exposure during puberty was associated with increased sperm concentration and motility but lower estradiol and higher FSH. The higher FSH triggered by low estradiol may be responsible for the increased sperm concentra- tion. Exposure during adulthood was not associated with any differences in semen characteristics or hormone level concentrations but the data were not shown. The small number of men exposed in adulthood (20) limited the power to detect ef- fects of exposure that took place in adulthood. Dhooge et al. (2006) studied Belgian men several months after a 1999 con- tamination of the food chain with PCBs and dioxins. Men were randomly selected from population registries, and 30% agreed to participate. Semen samples and serum for determination of dioxin-like activity (CALUX assay) and reproductive- hormone concentrations were collected from 101 men. Information on age, BMI, abstinence time, and other demographic characteristics was collected and used to adjust for possible confounding. No association was found between serum dioxin-like activity and total sperm count, sperm morphology, or serum gonado- tropins (FSH and LH). However, increasing serum-dioxin activity was associated with decreased semen volume (p = 0.02) and increased sperm concentration (p = 0.02). Increasing serum dioxin was also associated with decreasing testosterone. That association was of borderline statistical significance (p = 0.07) but became stronger (p = 0.04) when men with dioxin concentrations below 16 pg/L were ex- cluded (n = 11). The cut point of 16 pg/L was near the assayâs limit of detection, and readings in that range are subject to greater measurement error. The results were adjusted for age, BMI, and sampling date. Polsky et al. (2007) conducted a caseâcontrol study of patients seen in urology practices in Ontario, Canada. Eligible participants were 50â80 years old, had normal prostate-specific antigen concentrations, were not taking hor- monal medication, and had not received a diagnosis of prostate cancer. Of 335 eligible participants, 101 had a diagnosis of erectile dysfunction. The remain- ing 234 men served as controls. Controls had a variety of diagnoses of benign urologic conditions, including prostatic hyperplasia and urinary tract infections. Participants completed a questionnaire and had blood drawn for determination
452 VETERANS AND AGENT ORANGE: UPDATE 2008 of concentrations of specific PCB congeners. Cases and controls did not differ in concentrations of total PCBs or of any of the 14 congeners examined (regardless of adjustment for lipids and potential confounders). Toft et al. (2007) obtained semen and blood samples from 319 men in Po- land, Ukraine, Greenland, and Sweden. Participants were recruited differently in different locations. Dioxin-like activity was measured with the CALUX assay. No consistent association was found between dioxin-like activity and semen char- acteristics. In Poland, dioxin-like activity was associated with increased sperm concentration but not sperm motility or sperm count. Cok et al. (2008) collected adipose tissue samples from men who were un- dergoing abdominal surgical procedures (such as appendectomy) and compared the samples from infertile azoospermic men of normal karyotype with samples obtained from fertile men. They compared the concentrations of 29 congeners of dioxins, furans, and dioxin-like PCBs. The concentrations of two furans, 2,3,7,8- TCDF and 1,2,3,4,6,7,8,9-OCDF, were significantly higher in the infertile men than in the fertile men. However, the overall TEQ was significantly lower in the infertile men than in the fertile men (9.4 vs 12.5 pg/g); this difference remained statistically significant after controlling for age, BMI, smoking, and alcohol consumption. Female Fertility No new Vietnam-veteran or occupational studies addressing female fertility have been published since Update 2006. Environmental Studiesâ Eskenazi et al. (2007) and Warner et al. (2007) studied women in the Seveso Womenâs Health Study. The study identified women who were up to 40 years old at the time of the dioxin explosion in 1976. Women who lived in the most contaminated zones (A and B) and had adequate stored serum were enrolled during 1996â1998. For the Eskanazi et al. study of fibroids, women who had a diagnosis of fibroids before 1976 were excluded; this left a total of 956 women for analyses. Fibroids were ascertained by self-report, medical re- cords, and ultrasonographic examinations (634 women). The age-adjusted risk of fibroids was significantly lower in the high-exposure group (hazard ratio [HR] = 0.62, 95% CI 0.44â0.89) and in the middle-exposure group (HR = 0.58, 95% CI 0.41â0.81) than in the low-exposure group. The lower risk with higher exposure was not confounded by parity, family history of fibroids, age at menarche, current BMI, smoking, alcohol consumption, or education. In a study of menstrual function (Warner et al., 2007), women who were 20â40 years old and not taking oral contraceptives were evaluated with ultraso- nography (96 women), according to serum hormone concentrations (87), and ac- cording to the occurrence of ovulation (203). After adjustment for age and quality of the ultrasound, TCDD was not associated with the number or size of ovarian follicles, the occurrence of ovulation, or serum estradiol or progesterone.
REPRODUCTIVE EFFECTS AND IMPACTS ON FUTURE GENERATIONS 453 Chao et al. (2007) determined the concentrations of dioxins, furans, and PCBs in the placentas of 119 Taiwanese women. Dioxin TEQ, but not PCB TEQ, was associated with greater age at attaining âregular menstrual cycleâ whereas PCB TEQ, but not dioxin TEQ, was associated with greater âlength of longest menstrual cycle.â Those measures do not coincide exactly with the more common variables âage at first menstrual cycleâ and âlength of menstrual cycle,â which were examined and found not to be associated with dioxin exposure. The analyses and data presentation in the publication were unclear. Biologic Plausibility There is little evidence that 2,4-D or 2,4,5-T has substantial effects on re- productive organs or fertility. In contrast, many diverse laboratory studies have provided evidence that TCDD can affect reproductive-organ function and reduce fertility in both males and females. The administration of TCDD to male animals elicits reproductive toxicity by affecting testicular and seminal vesicle weight and function and by decreasing the rate of sperm production. The mechanisms of those effects are not known, but a primary hypothesis is that they are mediated through dysregulation of tes- ticular steroidogenesis. Exposure to TCDD is associated with increased estradiol secretion and decreased testosterone secretion; both hormones regulate sperm production. Both the AHR and the ARNT are expressed in rat and human testes, and studies suggest that TCDD causes tissue damage by inducing oxidative stress. Prostate cells are also responsive to TCDDâs inducting expression of various genes, including those involved in drug metabolism. Studies published since Update 2006 have reinforced those findings. Single intraperitoneal injections of TCDD induced marked histological changes in the testis, impaired spermato- genesis, increased serum estradiol, and decreased testosterone in male rats (Choi JS et al., 2008; Park JS et al., 2008). The effects of TCDD on the reproductive system of the male rat are critically dependent on the developmental time of exposure; for example, when fetal male rats were exposed to a single dose on gestational day 15, no decrease in sperm counts was observed although other developmental effects were found (Bell et al., 2007b). Many studies have examined the effects of TCDD on the female reproductive system. Two primary mechanisms that probably contribute to abnormal follicle development and decreased numbers of ova after TCDD exposure are cross-talk of the AHR with the estrogen receptor and dysregulation of the hypothalamic- pituitary-gonadal axis. Interaction of the activated AHR leads to inhibition of estradiol-induced gene expression and to enhancement of estrogen-receptor pro- tein degradation; both activities may contribute to TCDDâs antiestrogenic effects. TCDD also dysregulates the secretion pattern of preovulatory gonadotropin hor- mones, and this leads to abnormal and reduced follicle development. In addition, oocytes are directly responsive to TCDD. Thus, TCDDâs effects on hormone concentrations, hormone-receptor signaling, and ovarian responsiveness to hor-
454 VETERANS AND AGENT ORANGE: UPDATE 2008 mones all probably contribute to TCDD-induced female reproductive toxicity. Wang et al. (2006) showed that dioxins and dibenzofurans significantly correlate with dysregulation of estrogen metabolism in pregnant women. The reproductive organs of female experimental animals are targets for the action of TCDD (IOM, 2007). The ovary expresses both the AHR and the ARNT and is responsive to TCDD-inducible CYP1A1 and 1B1 expression, which de- pends on the phase of the estrous cycle (IOM, 2005). TCDD alters ovarian ste- roidogenesis, reducing ovarian expression of LH and FSH receptors, reducing circulating progesterone and estradiol, and decreasing fertility. TCDD decreases uterine weight, alters endometrial structure, and blocks estrogen-mediated en- dometrial proliferation and hypertrophy via an AHR-dependent mechanism in rodents and increases the incidence of endometriosis in rhesus monkeys. Since Update 2006, additional work addressing TCDDâs effects on female reproduction in animal models has been published. The data of Heiden et al. (2008) on zebrafish suggest that TCDD inhibits follicle maturation via attenu- ated gonadotropin responsiveness or depression of estradiol biosynthesis and that interference of estrogen-regulated signal transduction may also contribute to TCDDâs effects on follicular development, possibly by disrupting signaling pathways, such as glucose and lipid metabolism, and disrupting regulation of transcription. Exposure of cultured luteinized human granulose cells to TCDD increased expression of inhibin A, which would be expected to produce a reduction in FSH-stimulatable estrogen secretion (Ho et al., 2006). The AHR has been found to be required for normal ovulation and gonadotropin responses in the mouse ovary, as shown in AHR-null mice, which have lower concentrations of LH-re- ceptor and FSH-receptor expression than their wild-type counterparts. AHR-null mice produce fewer eggs than wild-type mice in response to human chorionic gonadotropin; this, suggests that their follicles have a lower capacity to ovulate than do follicles of wild-type mice because of reduced responsiveness to go- nadotropins (Barnett et al., 2007a). Barnett et al. (2007b) had previously shown that the follicles of AHR-null mice grew more slowly than follicles of wild-type mice but that administration of estradiol restored the normal growth rate, per- haps because of the AHR affected follicular growth via mechanisms that involve estradiol regulation and responsiveness. Similarly, Ye and Leung (2008) showed that TCDD down-regulated expression of the CYP19 gene, which codes for the aromatase enzyme responsible for the conversion of androstenedione to estrone and of testosterone to estradiol, indicating that exposure to TCDD was directly antiestrogenic by decreasing estrogen. Ovarian endocrine disruption appears to be the predominant functional change induced by chronic exposure to the low doses of TCDD that are associated with premature reproductive senescence in female rats without depletion of ovarian follicular reserves (Shi et al., 2007). TCDD exhibits antiestrogenic properties, including antiuterotrophic effects (such as inhibition of estrogen-induced uterine growth and proliferation), possi-
REPRODUCTIVE EFFECTS AND IMPACTS ON FUTURE GENERATIONS 455 bly by inhibiting estrogen-mediated gene expression through estrogen-receptor/ AHR cross-talk. The effects of TCDD on changes in the expression of uterine genes mediated by ethynyl estradiol (EE) were investigated by using cDNA mi- croarrays and physiologic and histologic determinations. EE-TCDD cotreatment inhibited the increase in uterine wet weight and the reductions in stromal edema, hypertrophy, and hyperplasia induced by EE alone. The cotreatment also induced marked luminal epithelial-cell apoptosis. Of the 2,753 EE-mediated differentially expressed genes, only 133 were significantly modulated by TCDD cotreatment; the modulation might be because of a gene-specific inhibitory response. The EE- mediated induction of several genes, including trefoil factor 1 and keratin 14, was inhibited by more than 90% by TCDD. Comparison with public databases found that the genes whose expression was inhibited are known to have functions asso- ciated with cell proliferation, water and ion transport, and maintenance of cellular structure and integrity; this theoretical profile of functional impacts is consistent with the observed histological alterations and sheds light on possible mechanisms for TCDDâs antiuterotrophic effects (Boverhof et al., 2008). Although it would not constitute an adverse health outcome in an individual veteran, there is fairly strong evidence (see Table 7-4) that paternal exposure to dioxin may result in a lower sex ratio (that is, a smaller than expected proportion of male infants at birth). Pronounced reductions in sex ratio have been observed in the offspring of men exposed to dioxin after the Seveso accident, especially those under 19 years old at the time of the dioxin release (Mocarelli et al., 2000); this phenomenon was not observed in the offspring of young women exposed by the Seveso accident (Baccarelli et al., 2008). Similar results of a depression in the sex ratio concentrated among fathers who were under 20 years old at the time of the incident were following the Yucheng poisoning with oil contaminated with PCBs, PCDFs, and PCDDs (del Rio Gomez et al., 2002). Reductions in the expected number of male offspring have also been reported in several cohorts of men occupationally exposed to dioxin (Moshammer and Neuberger, 2000; Ryan et al., 2002), but other such cohorts did not manifest this relationship (Heacock et al., 1998; Savitz et al., 1997; Schnorr et al., 2001). In the single report relevant to this outcome in Vietnam veterans, however, the sex ratio was increased in the Ranch Hand group that had the highest serum dioxin concentrations (Michalek et al., 1998b). There were three articles published after Update 2006 that contributed in- formation on whether TCDD exposure alters the sex ratio. Chao et al. (2007) mention that they did not find an association between sex ratio of the offspring and the TEQ concentrations of dioxins, furans, or PCBs in the placentas from 119 Taiwanese women. Crude sex ratios for all births in 1994â2005 to women who were less than 18 years old at the time of the Seveso accident are reported in Baccarelli et al. (2008), and the proportion of male births exceeds that of female births in Zones A and B. The only new evidence of an effect on sex ratio came from Hertz-Picciotto et al. (2008), who reported on serum concentrations of nine
456 VETERANS AND AGENT ORANGE: UPDATE 2008 TABLE 7-4â Selected Epidemiologic StudiesâSex Ratioa Sex Ratio of Offspring Reference Study Population (boys/total)b Comments VIETNAM VETERANS Studies Reviewed in Update 2002 Michalek Births from service through 1993 in et al., 1998b AFHS Comparison group 0.504 Not formally analyzed Dioxin level in Ranch Hand personnel Background 0.502 Low 0.487 High 0.535 OCCUPATIONAL Studies Reviewed in Update 2004 Ryan et al., Russian workers manufacturing 2,4,5- 0.401 p < 0.001, either 2002 trichlorophenol (1961â1988) or 2,4,5-T (91 boys: 136 girls) parent exposed (1964â1967) 0.378 p < 0.001, only (71 boys: 117 girls) father exposed 0.513 ns, only mother (20 boys: 19 girls) exposed Studies Reviewed in Update 2002 Schnorr Workers producing trichlorophenol and No difference on et al., 2001 derivatives, including 2,4,5-T basis of age at Serum TCDD in fathers first exposure Neighborhood controls (< 20 ppt) 0.544 Referent Worker fathers < 20 ppt 0.507 None 20â255 ppt 0.567 significantly 255â < 1,120 ppt 0.568 decreased (or â¥ 1,120 ppt 0.550 increased) Moshammer Austrian chloracne cohort Fewer sons, and Children born after starting TCDD 0.464 especially if Neuberger, exposure in 1971 (26 boys: 30 girls) father was under 2000 Children born before 1971 0.613 20 years old (19 boys: 12 girls) when exposed: SR = 0.20 (1 boy: 4 girls) Savitz et al., OFFHS fathersâ exposure during 3 mo 1997 before conception: No chemical activity 0.503 Referent Crop herbicides (some phenoxy herbicides) 0.500 ns Protective equipment used not used 0.510 ns No protective equipment 0.450 ns Studies Reviewed in Update 1998 Heacock Sawmill workers in British Columbia et al., 1998 Chlorophenate-exposed workers 0.515 Nonexposed workers 0.519 Province overall 0.512
REPRODUCTIVE EFFECTS AND IMPACTS ON FUTURE GENERATIONS 457 TABLE 7-4â Continued Sex Ratio of Offspring Reference Study Population (boys/total)b Comments ENVIRONMENTAL New Studies Baccarelli Births 1994â2005 in women 0â28 years et al., 2008 old at time of Seveso accident Zone A 0.571 Zone B 0.508 Zone R 0.495 Hertz- San Francisco Bay areaâserum OR for male birth SRs all < 0.5 Picciotto concentrations in pregnant women during (not SR) et al., 2008 1960s 90th percentile vs 10th percentile Total PCBs 0.4 (0.3â0.8) p = 0.007 dl PCBs PCB 105 0.6 (0.4â0.9) p = 0.02 PCB 118 0.7 (0.5â1.2) p = 0.17 PCB 170 0.6 (0.4â0.9) p = 0.02 PCB 180 0.8 (0.5â1.2) p = 0.32 Chao et al., Taiwanâplacental TEQ concentrations of 2007 TCDDs, TCDFs, PCBs nr No association del Rio Births in individuals exposed to PCBs, vs unexposed Gomez PCDFs, PCDDs in 1979 Yucheng incident with same et al., 2002 demographics Father exposed (whether or not mother exposed) 0.490 p = 0.037 Father under 20 years old in 1979 0.458 p = 0.020 Father at least 20 years old in 1979 0.541 p = 0.60 Mother exposed (whether or not father exposed) 0.504 p = 0.45 Mother under 20 years old in 1979 0.501 p = 0.16 Mother at least 20 years old in 1979 0.500 p = 0.40 Studies Reviewed in Update 2002 Karmaus Births after 1963 to Michigan fish-eaters et al., 2002 with serum PCBs in both parents Paternal PCBs > 8.1 Âµg/L 0.571 p < 0.05 (but for more sons) Maternal PCBs > 8.1 Âµg/L 0.494 ns Yoshimura Parents (one or both) exposed to PCBs, et al., 2001 PCDFs in Yusho, Japan All Japan in 1967 0.513 Referent Births 1967 (before poisoning incident) 0.516 ns Births 1968â1971 (after incident) 0.574 ns continued
458 VETERANS AND AGENT ORANGE: UPDATE 2008 TABLE 7-4â Continued Sex Ratio of Offspring Reference Study Population (boys/total)b Comments Revich Residents near chemical plant in operation et al., 2001 1967â1987 in Chapaevsk, Russia 1983â1997 0.507 No clear pattern Minimumâin 1989 0.401 Maximums in 1987 0.564 in 1995 0.559 Studies Reviewed in Update 2000 Mocarelli Births 1977â1996 in people from Zones et al., 2000 A, B, R, 3â45 years old at time of 1976 Seveso accident 0.514 Referent Neither parent exposed 0.608 ns Father exposed (whether or not mother exposed) 0.440 p = 0.03 Father under 19 years old in 1976 0.382 p = 0.002 Father at least 19 years old in 1976 0.469 ns Only mother exposed 0.545 ns Studies Reviewed in Update 1998 Mocarelli Parent (either sex) from Seveso Zone A et al., 1996 Births 1977â1984 0.351 p < 0.001, related (26 boys: 48 girls) to parental TCDD serum Births 1985â1994 0.484 ns (60 boys: 64 girls) ABBREVIATIONS: 2,4,5-T, 2,4,5-trichlorophenoxyacetic acid; AFHS, Air Force Health Study; dl, dioxin-like; ns, not significant; nr, not reported; OFFHS, Ontario Farm Family Health Study; OR, odds ratio; PCB, polychlorinated biphenyl; PCDD, polychlorinated dibenzodioxin; PCDF, polychlo- rinated dibenzofurans; SR, sex ratio; TCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxin; TCDF, tetrachlo- rodibenzofuran; TEQ, toxicity equivalent quotient. aVAO reports before Update 1998 did not address association between perturbations in sex ratio of offspring and exposure to chemicals of interest. bGiven when available. PCB congeners (four of which were dioxin-like: PCB 105, 118, 170, and 180) in blood gathered during the 1960s from 399 pregnant women in the San Francisco Bay area. The adjusted odds of a male birth were significantly decreased when the 90th percentile of the total concentration of all nine PCBs was compared with the 10th percentile (OR = 0.45, 95% CI 0.26â0.80). The proportion of male births was significantly reduced for two of the dioxin-like PCBs analyzed separately, and the decrease in the proportion of male babies was not significant for any of the five nonâdioxin-like PCBs. A population-level finding of a paternally mediated effect would be a strong indicator that dioxin exposure can interfere with the male reproductive process.
REPRODUCTIVE EFFECTS AND IMPACTS ON FUTURE GENERATIONS 459 To date, however, the results for a reduced number of sons for exposed fathers are mixed. James (2006) has interpreted perturbation of sex ratios by dioxins and other agents as being an indicator of parental endocrine disruption. If Jamesâ hypothesis were demonstrated to hold, it would be concordant with observing a reduction in testosterone levels among exposed men. Another pathway to an altered sex ratio might involve male embryos experiencing more lethality with induction of mutations due to their unmatched X chromosome. A genotoxic mechanism has not been expected to apply to TCDD, but gender-specific adverse consequences of modified imprinting of gametes might be a possible mechanism leading to observation of altered sex ratios at birth. There has been no work with experimental animals that specifically exam- ines the effects of TCDD on sex ratios of offspring, nor have any alterations in sex ratio been reported in animal studies that examined developmental effects of TCDD on offspring. Synthesis Reproduction is sensitive to TCDD and DLCs in rodents. It is clear that the fetal rodent is more sensitive to adverse effects of TCDD than the adult rodent. The sensitivity in humans is less apparent. There is little evidence that exposure to dioxin is associated with a reduction in sperm quality or a reduction in fertil- ity. However, the committee notes that the evidence that TCDD exposure reduces serum testosterone in men is consistent among several epidemiologic studies with appropriate consideration of confounders, including one of Vietnam veterans; shows a doseâresponse relationship; and is biologically plausible on the basis of concomitant increases observed in gonadotropins and biologic plausibility shown by animal studies. Human populations showing evidence of reduced testosterone with exposure to DLCs include a general population sample (Dhooge et al., 2006), occupationally exposed people (Egeland et al., 1994), and Vietnam veter- ans in the Air Force Health Study (Gupta et al., 2006). The evidence that DLCs may modify the sex ratio lends credence to the hypothesis that these chemicals affect male reproductive functioning. Despite the general consistency of the findings of a reduction in testosterone concentration, the testosterone concentrations observed even in the most exposed groups studied are well within the normal range. The small reduction in testoster- one is not expected to have adverse clinical consequences. There is evidence that compensatory physiologic mechanisms come into play. The occupational study of Egeland et al. (1994) found increased gonadotropins in addition to reduced testos- terone. The gonadotropins stimulate the production of testosterone in men. Conclusions On the basis of the evidence reviewed here and in previous VAO reports, the committee concludes that there is inadequate or insufficient evidence of an
460 VETERANS AND AGENT ORANGE: UPDATE 2008 association between exposure to the chemicals of interest and decreased sperm counts or sperm quality, subfertility, or infertility. SPONTANEOUS ABORTION Spontaneous abortion is the expulsion of a nonviable fetus, generally before 20 weeks of gestation, that is not induced by physical or pharmacologic means. The background risk of recognized spontaneous abortion is generally 7â15% (Hertz-Picciotto and Samuels, 1988), but it is established that many more preg- nancies terminate before women become aware of them (Wilcox et al., 1988); such terminations are known as subclinical pregnancy losses and generally are not included in studies of spontaneous abortion. Estimates of the risk of recog- nized spontaneous abortion vary with the design and method of analysis. Studies have included cohorts of women asked retrospectively about pregnancy history, cohorts of pregnant women (usually those receiving prenatal care), and cohorts of women who are monitored for future pregnancies. The value of retrospective reports can be limited by memory loss, particularly of spontaneous abortions that took place long before. Studies that enroll women who appear for prenatal care require the use of life tables and specialized statistical techniques to account for differences in the times at which women seek medical care during pregnancy. Enrollment of women before pregnancy provides the theoretically most valid estimate of risk, but it can attract nonrepresentative study groups because proto- cols are demanding. Conclusions from VAO and Updates The committee responsible for the original VAO report concluded that there was inadequate or insufficient evidence of an association between exposure to 2,4-D, 2,4,5-T, TCDD, picloram, or cacodylic acid and spontaneous abortion. Additional information available to the committees responsible for Update 1996, Update 1998, and Update 2000 did not change that conclusion. The committee responsible for Update 2002, however, did conclude that there was enough evidence available concerning paternal exposure to TCDD specifically to conclude that there was suggestive evidence that paternal exposure to TCDD is not associated with the risk of spontaneous abortion. This convic- tion was based primarily on the National Institute for Occupational Safety and Health (NIOSH) study (Schnorr et al., 2001), which investigated a large number of pregnancies fathered by workers whose serum TCDD levels were extrapolated back to the time of conception; no association was observed up to the highest exposure group (â¥ 1,120 ppt). Indications of positive association were seen in studies of Vietnam veterans (CDC, 1989; Field and Kerr, 1988; Stellman et al., 1988), but the committee for Update 2002 asserted that they might be due to exposure to phenoxy herbicides rather than to TCDD and concluded that there
REPRODUCTIVE EFFECTS AND IMPACTS ON FUTURE GENERATIONS 461 was insufficient information to determine whether an association exists between maternal exposure to TCDD and the risk of spontaneous abortion or between maternal or paternal exposure to 2,4-D, 2,4,5-T, picloram, or cacodylic acid and the risk of spontaneous abortion. The additional information (none of which concerned paternal exposures) re- viewed by the committees responsible for Update 2004 and Update 2006 did not change that conclusion. The relevant studies are reviewed in the earlier reports. Table 7-5 summarizes their findings. Update of the Epidemiologic Literature No occupational or veterans studies on spontaneous abortion in relation to the chemicals of interest have been published since Update 2006. Tsukimori et al. (2008) gathered the reproductive history back to 1958 of 214 women involved in the 1968 Yusho incident of ingestion of rice oil contaminated with PCBs and other dioxin-like compounds. Information was gathered on 512 pregnancies: 204 in 1958â1967 served as the referent group, 122 in the 10 years after the incident (1968â1977) were of primary interest, and 88 in 1978â1987 and 98 in 1988â2003 were used to show the degree to which adverse reproduc- tive effects had abated. Blood samples were drawn from 97 of the women in 2001â2005; analyses focused on three DLCs: 2,3,4,7,8-pentachlorodibenzofuran (PeCDF), PCB 126 (total equivalency factor [TEF] = 0.1), and PCB 169 (TEF = 0.01). The risk of spontaneous abortion in all pregnancies (excluding induced abortions) in the 10 years after the incident was higher than the risk before it (OR = 2.09, 95% CI 0.84â5.18) but fell to more neutral values in 1978â1987 (OR = 1.00, 95% CI 0.32â3.09) and 1988â2003 (OR = 1.22, 95% CI 0.41â3.63). When the pregnancies of the women whose serum was reviewed for the DLCs were analyzed, an increased risk of spontaneous abortion were found for 10- fold increases in serum concentrations of all three: PeCDF (OR = 1.60, 95% CI 1.10â2.33), PCB 126 (OR = 2.52, 95% CI 0.92â6.87), and PCB 169 (OR = 2.28, 95% CI 1.09â4.75). In studying pregnant Taiwanese women, Chao et al. (2007) mentioned that they did not find an association between placental dioxin, PCB TEQ, or indicator PCBs and spontaneous abortion, but the data were not presented. Biologic Plausibility Laboratory animal studies have demonstrate that TCDD exposure during pregnancy can alter concentrations of circulating steroid hormones and disrupt placental development and function and thus contribute to a reduction in survival of implanted embryos and to fetal death. However, the reproductive significance of those effects and the risk of recognized pregnancy loss before 20 weeks of gestation in humans are not clear. There is no evidence of a relationship between
462 VETERANS AND AGENT ORANGE: UPDATE 2008 TABLE 7-5â Selected Epidemiologic StudiesâSpontaneous Abortiona Exposed Estimated Relative Reference Study Population Casesb Risk (95% CI)b VIETNAM VETERANS Studies Reviewed in Update 2002 Kang et al., Female Vietnam-era veterans (maternal exposure) 1.0 (0.82â1.21) 2000 Vietnam veterans (1,665 pregnancies) 278 nr Vietnam-era veterans who did not serve in Vietnam (1,912 pregnancies) 317 nr Studies Reviewed in Update 2000 Schwartz, Female Vietnam veterans (maternal exposure) 1998 Women who served in Vietnam 113 nr Women who did not serve in the war zone 124 nr Civilian women 86 nr Studies Reviewed in Update 1996 Wolfe Air Force Ranch Hand veterans 157 et al., 1995 Background 57 1.1 (0.8â1.5) Low exposure 56 1.3 (1.0â1.7) High exposure 44 1.0 (0.7â1.3) Studies Reviewed in VAO Aschengrau Wives of Vietnam veterans presenting at Boston and Hospital for Women Monson, 27 weeks of gestation 10 0.9 (0.4â1.9) 1989 13 weeks of gestation nr 1.2 (0.6â2.8) CDC, 1989 Vietnam Experience Study Overall 1,566 1.3 (1.2â1.4) Self-reported low exposure 489 1.2 (1.0â1.4) Self-reported medium exposure 406 1.4 (1.2â1.6) Self-reported high exposure 113 1.7 (1.3â2.1) Field and Follow-up of Australian Vietnam veterans 199 1.6 (1.3â2.0) Kerr, 1988 Stellman American Legionnaires with service 1961â1975 et al., 1988 Vietnam veterans vs Vietnam-era veterans All Vietnam veterans 231 1.4 (1.1â1.6) Low exposure 72 1.3 (1.0â1.7) Medium exposure 53 1.5 (1.1â2.1) High exposure 58 1.7 (1.2â2.4) Vietnam-era veterans vs herbicide handlers 9 1.6 (0.7â3.3) Vietnam veterans Low exposure 72 1.0 Medium exposure 53 1.2 (0.8â1.7) High exposure 58 1.4 (0.9â1.9) OCCUPATIONAL Studies Reviewed in Update 2002 Schnorr Wives and partners of men in NIOSH cohort et al., 2001 Estimated paternal TCDD serum at time of conception < 20 ppt 29 0.8 (0.5â1.2) 20 to < 255 ppt 11 0.8 (0.4â1.6) 255 to < 1120 11 0.7 (0.3â1.6) â¥ 1120 ppt 8 1.0 (0.4â2.2)
REPRODUCTIVE EFFECTS AND IMPACTS ON FUTURE GENERATIONS 463 TABLE 7-5â Continued Exposed Estimated Relative Reference Study Population Casesb Risk (95% CI)b Studies Reviewed in Update 2000 Driscoll, Women employed by US Forest Serviceâ 1998 miscarriages (maternal exposure) 141 2.0 (1.1â3.5) Studies Reviewed in VAO Moses Follow-up of 2,4,5-T production workers et al., 1984 14 0.9 (0.4â1.8) Suskind Follow-up of 2,4,5-T production workers 69 0.9 (0.6â1.2) and Hertzberg, 1984 Smith Follow-up of 2,4,5-T sprayers vs nonsprayers 43 0.9 (0.6â1.3)c et al., 1982 Townsend Wives of men employed involved in chlorophenol et al., 1982 processing at Dow Chemical Co. 85 1.0 (0.8â1.4) Carmelli Wives of men occupationally exposed to 2,4-D et al., 1981 reported work exposure to herbicides (high All and medium) 63 0.8 (0.6Ââ1.1)c Farm exposure 32 0.7 (0.4â1.5)c Forest and commercial exposure 31 0.9 (0.6â1.4)c Exposure during conception period Farm exposure 15 1.0 (0.5â1.8)c Forest and commercial exposure 16 1.6 (0.9â1.8)c Fathers 18â25 years old Farm exposure 1 0.7 (nr) Forest and commercial exposure 3 4.3 (nr) Fathers 26â30 years old Farm exposure 4 0.4 (nr) Forest and commercial exposure 8 1.6 (nr) Fathers 31â35 years old Farm exposure 10 2.9 (nr) Forest and commercial exposure 5 1.0 (nr) ENVIRONMENTAL New Studies Chao et al., Pregnant Taiwanese women, placental TEQ of 2007 dioxins, PCBs (maternal exposure) nr, but reported ns Tsukimori Spontaneous abortions among pregnancies et al., 2008 (excluding induced abortions) of women in 1968 Yusho incident (maternal exposure) 10 years after vs 10 years before nr 2.1 (0.8â5.2) 10-fold increase in maternal blood concentration (drawn 2001â2005) of: PeCDF nr 1.6 (1.1â2.3) PCBâ126 (TEF = 0.1) nr 2.5 (0.9â6.9) PCBâ169 (TEF = 0.01) nr 2.3 (1.1â4.8) continued
464 VETERANS AND AGENT ORANGE: UPDATE 2008 TABLE 7-5â Continued Exposed Estimated Relative Reference Study Population Casesb Risk (95% CI)b Studies Reviewed in Update 2006 Eskenazi SWHS participants living in exposure Zones A, B et al., 2003 in 1976 (maternal exposure) Pregnancies 1976â1998 97 0.8 (0.6â1.2) Pregnancies 1976â1984 44 1.0 (0.6â1.6) Studies Reviewed in Update 2002 Arbuckle Ontario farm families (maternal and paternal et al., 2001 exposure) Phenoxyacetic acid herbicide exposure in preconception period, spontaneous-abortion risk 48 1.5 (1.1â2.1) Revich Residents of Samara Region, Russia (maternal et al., 2001 and paternal exposure) Chapaevsk nr 24.4% (20.0â29.5%)d Samara nr 15.2% (14.3â16.1%)d Toliatti nr 10.6% (9.8â11.5%)d Syzran nr 15.6% (13.4â18.1%)d Novokuibyshevsk nr 16.9% (14.0â20.3%)d Other small towns nr 11.3% (9.4â13.8%)d Tuyet and Vietnamese women who were or whose husbands nr nr, anecdotal reports Johansson, were exposed to herbicides sprayed during of miscarriage in 2001 Vietnam War pilot study ABBREVIATIONS: 2,4Â-D, 2,4-dichlorophenoxyacetic acid; 2,4,5-T, 2,4,5-trichlorophenoxyacetic acid; CI, confidence interval; NIOSH, National Institute for Occupational Safety and Health; nr, not reported; ns, not significant (usually refers to p < 0.05); PCB, polychlorinated biphenyl; PeCDF, 2,3,4,7,8- p Â entachlorodibenzofuran; SWHS, Seveso Womenâs Health Study; TCDD, 2,3,7,8-tetrachlorodibenzo- p-dioxin; TEF, toxic equivalency factor; TEQ, toxicity equivalent quotient. aUnless otherwise indicated, results are for paternal exposure. bGiven when available; results other than estimated risk explained individually. c90% CI. dSpontaneous abortion rate per 100 full-term pregnancies for 1991â1997. paternal or maternal exposure to TCDD and spontaneous abortion. Exposure to 2,4-D or 2,4,5-T causes fetal toxicity and death after maternal exposure in experimental animals. However, that effect occurs only at high doses and in the presence of maternal toxicity. No fetal toxicity or death has been reported to oc- cur after paternal exposure to 2,4-D. Synthesis The two new environmental studies with information on DLCs and spontane- ous abortion had conflicting results and do not constitute motivation to change the assessment of the prior committees. Given the age of the Vietnam-veteran cohort,
REPRODUCTIVE EFFECTS AND IMPACTS ON FUTURE GENERATIONS 465 publication of additional information on this outcome in the target population of the VAO series is unlikely. Conclusions On the basis of the evidence reviewed here and in previous VAO reports, the committee concludes that paternal exposure to TCDD is not associated with risk of spontaneous abortion and that insufficient information is available to de- termine whether an association exists between the risk of spontaneous abortion and maternal exposure to TCDD or either maternal or paternal exposure to 2,4-D, 2,4,5-T, picloram, or cacodylic acid. STILLBIRTH, NEONATAL DEATH, AND INFANT DEATH Stillbirth or late fetal death typically refers to the delivery at or after 20 weeks of gestation of a fetus that shows no signs of life, including fetuses that weigh more than 500 g regardless of gestational age (Kline et al., 1989). Neonatal death refers to the death of a liveborn infant within 28 days of birth, and infant death includes deaths occurring before the first birthday. Because the causes of stillbirth and early neonatal death overlap consider- ably, they are commonly analyzed together in a category referred to as perinatal mortality (Kallen, 1988). Stillbirths make up less than 1% of all births (CDC, 2000). The most common causes of perinatal mortality (Kallen, 1988) among low-birth-weight (500â2,500 g) liveborn and stillborn infants are placental and delivery complicationsâabruptio placenta, placenta previa, malpresentation, and umbilical-cord conditions. The most common causes of perinatal death of infants weighing more than 2,500 g at birth are complications of the cord, placenta, and membranes and congenital malformations (Kallen, 1988). Conclusions from VAO and Updates The committee responsible for VAO concluded that there was inadequate or insufficient evidence of an association between exposure to 2,4-D, 2,4,5-T, TCDD, picloram, or cacodylic acid and stillbirth, neonatal death, or infant death. Additional information available to the committees responsible for Update 1996, Update 1998, Update 2000, Update 2002, Update 2004, and Update 2006 did not change that conclusion. Reviews of the relevant studies are presented in the earlier reports. Update of the Epidemiologic Literature No additional occupational or veterans studies on perinatal death in relation to the chemicals of interest have been published since Update 2006.
466 VETERANS AND AGENT ORANGE: UPDATE 2008 In the study of pregnancy results in women involved in the Yusho poison- ing incident, Tsukimori et al. (2008) reported on the intensity of pregnancy loss, defined as the risk of stillbirth, neonatal death, or spontaneous abortion among all pregnancies after induced abortions had been excluded. The risk of pregnancy loss was higher in the 10 years after the incident than before the incident period (OR = 2.11, 95% CI 0.92â4.87) but fell to more neutral values in 1978â1987 (OR = 1.02, 95% CI 0.37â2.84) and in 1988â2003 (OR = 1.01, 95% CI 0.37â2.78). In women whose serum concentrations of the three DLCs had been measured, the risk of pregnancy loss increased with 10-fold increases in serum concentrations of each: PeCDF (OR = 1.70, 95% CI 1.18â2.46), PCB 126 (OR = 2.99, 95% CI 1.16â7.73), and PCB 169 (OR = 2.68, 95% CI 1.32â5.48). Inclusion of stillbirths and neonatal deaths made the risk higher than that of spontaneous abortions alone (see above), but spontaneous abortions appear to dominate the analyses. Biologic Plausibility Laboratory studies of maternal TCDD exposure during pregnancy have demonstrated the induction of fetal death; neonatal death, however, is only rarely observed and is usually the result of cleft palate, which leads to an inability to nurse. Studies addressing the potential for perinatal death as a result of paternal exposure to TCDD or herbicides are inadequate to support conclusions. One new study (Kransler et al., 2008) evaluated fetal and neonatal viability in rats exposed to various TCDD doses. Mortality in exposed rats was very high at gestation day 20 and postnatal day 7, but the causes of death were not determined in any of the cases. Synthesis The Yusho study reviewed for this update did find significant associations between maternal serum concentrations of three DLCs and rates of pregnancy loss, a variable that included stillbirth and neonatal death but was dominated by spontaneous abortion. The committee, however, considers studies of exposures to dioxin-like PCBs and furans as supportive studies of dioxin rather than present- ing primary evidence. Given the age of the Vietnam-veteran cohort, publication of additional information on this outcome in the target population of the VAO series is highly unlikely. Conclusions On the basis of the evidence reviewed here and in previous VAO reports, the committee concludes that there is inadequate or insufficient evidence of an association between exposure to the chemicals of interest and stillbirth, neonatal death, or infant death.
REPRODUCTIVE EFFECTS AND IMPACTS ON FUTURE GENERATIONS 467 BIRTH WEIGHT AND PRETERM DELIVERY Birth weight and the length of the gestation period can have significant ef- fects on neonatal morbidity and mortality. Defined by the World Health Organiza- tion as birth weights < 2,500 grams (Alberman, 1984), low birth weight is a health outcome resulting from one of two distinct causes. Intrauterine growth retardation (IUGR) occurs when fetal growth is diminished and a fetus or baby fails to attain a normal weight or is small for gestational age. The concept of IUGR represents birth weight adjusted for gestational age, resulting in a lower weight than average when compared to local or national fetal growth graphs (Romo et al., 2009). Low birth weight can also occur secondary to preterm delivery (PTD), which is deliv- ery at less than 259 days, or 37 completed weeks, of gestation, calculated on the basis of the date of the first day of the last menstrual period (Bryce, 1991). Low birth weight resulting from either of these causes occurs in approximately 7% of live births. When no distinction is made between the causes of low birth weight (IUGR or PTD), the factors most strongly associated with it are maternal tobacco use during pregnancy, multiple births, and race or ethnicity. Other potential risk factors are low socioeconomic status (SES), malnutrition, maternal weight, birth order, maternal complications during pregnancy (such as severe pre-eclampsia or intrauterine infections) and obstetric history, job stress, and cocaine or caffeine use during pregnancy (Alexander and Slay, 2002; Alexander et al., 2003; Ergaz et al., 2005; Kallen, 1988; Peltier, 2003). Established risk factors for PTD include race (black), marital status (single), low SES, previous low birth weight or PTD, multiple gestations, tobacco use, and cervical, uterine, or placental abnormalities (Berkowitz and Papiernik, 1993). Conclusions from VAO and Updates The committee responsible for VAO concluded that there was inadequate or insufficient evidence of an association between exposure to the chemicals of interest and low birth weight or PTD. Additional information available to the committees responsible for Update 1996, Update 1998, Update 2000, Update 2002, Update 2004, and Update 2006 did not change that conclusion. Reviews of the relevant studies are presented in the earlier reports. Update of the Epidemiologic Literature No new occupational or Vietnam-veteran studies concerning exposure to the chemicals of interest and low birth weight or PTD have been published since Update 2006. In reporting on neonatal thyroid function among the births in 1994â2005 to women who had been less than 28 years old at the time of the Seveso accident, Baccarelli et al. (2008) noted that birth weight was similar among the three expo-
468 VETERANS AND AGENT ORANGE: UPDATE 2008 sure zones. The data were not formally analyzed, but tabulated data indicated that the proportions of low-birth-weight infants (under 2,500 g) were not excessive: Zone A (1.8%, based on only 32 births), Zone B (5.2%), and Zone R (5.9%). In the study of pregnancy results in women involved in the Yusho poison- ing incident, Tsukimori et al. (2008) reported on the frequency of PTD. The risk of PTD in the 10 years after the incident was significantly higher than that before the incident (OR = 5.70, 95% CI 1.17â27.79) but was less pronounced in 1978â1987 (OR = 1.46, 95% CI 0.20â10.49) and in 1988â2003 (OR = 2.09, 95% CI 0.33â13.20). In the women whose serum concentrations of the three DLCs had been measured, the risk of PTD increased with 10-fold increases in serum concentrations of each: PeCDF (OR = 1.98, 95% CI 1.03â3.80), PCB 126 (OR = 4.90, 95% CI 0.93â25.75), and PCB 169 (OR = 4.12, 95% CI 1.19â14.30). Sagiv et al. (2007) measured PCB congeners in the cord blood of 788 in- fants born in 1993â1998 to mothers at least 18 years old and residing around the PCB-contaminated harbor in New Bedford, Massachusetts. After adjustment for infant gestational age, sex, and year of birth and for maternal age, race, parity, BMI, smoking, and fish consumption, tests for trends in birth weight, crown-to- heel length, and head circumference with cord serum concentrations of dioxin- like PCB 118 (TEF = 0.0001) or with the sum of mono-ortho TEFs were all nonsignificant. Similarly, Nishijo et al. (2008) studied 42 motherâinfant pairs born at Toyama University Hospital, Japan, after at least 30 weeks of gestation. They did not find significant correlations between TEQâtotal, TEQâPCDD, or TEQâPCDF in maternal breast milk and birth weight. There were also no patterns of correlation in these measures of dioxin-like activity and infant length, chest circumference, or head circumference at birth. Biologic Plausibility The available experimental evidence on animals indicates that TCDD expo- sure during pregnancy can reduce body weight at birth but only at high doses. Laboratory studies of the potential male-mediated developmental toxicity of TCDD and herbicides as a result of exposure of adult male animals are inadequate to permit conclusions. TCDD and herbicides are known to cross the placenta, and this leads to direct exposure of the fetus. Data from studies of experimental animals also suggest that the preimplantation embryo and developing fetus are sensitive to the toxic effects of 2,4-D and TCDD after maternal exposure. How- ever, the significance of those animal effects for humans is not clear. Synthesis The four environmental studies reviewed here did not provide compelling evidence of an association between exposure to the chemicals of interest and the risk of low birth weight or prematurity. The increased risk of PTD in the 5 years
REPRODUCTIVE EFFECTS AND IMPACTS ON FUTURE GENERATIONS 469 after the Yusho incident and specifically in relation to measures of dioxin-like activity (Tsukimori et al., 2008) is of interest, but as the authors note, their sample included fewer than half the eligible women and the pregnancy information was gathered from the subjects long after the events. Otherwise, the results overall suggest a lack of an association with various measures of in utero develop- ment. Given the age of the Vietnam-veteran cohort, publication of additional information on this outcome in the target population of the VAO series is highly unlikely. Conclusions On the basis of the evidence reviewed here and in previous VAO reports, the committee concludes that there is inadequate or insufficient evidence of an association between exposure to the compounds of interest and low birth weight or preterm delivery. BIRTH DEFECTS The March of Dimes defines a birth defect as an abnormality of structure, function, or metabolism, whether genetically determined or as the result of an en- vironmental 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 2â3% of live births, are abnormalities that are present at birth and are severe enough to interfere with viability or physical well- being. Birth defects are detected in another 5% of babies through the first year of life. The causes of most birth defects are unknown. Genetic factors, exposure to some medications, exposure to environmental contaminants, occupational expo- sures, 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 some work has addressed paternal exposures. Paternally mediated exposures might occur by several routes and exert effects in various ways. One way is through direct genetic damage to the male germ cell transmitted to the offspring and dominantly expressed as a birth defect. A hypothesized route is the transfer of toxic compounds through a manâs body into his seminal fluid, resulting in intermittent fetal exposures throughout gestation (Chia and Shi, 2002). Another, even more indirect route of paternally mediated exposure could be contact of family members with contamination brought into the home from the workplace, but this would not be applicable to offspring of Vietnam veterans conceived after deployment. Conclusions from VAO and Updates The committee responsible for VAO determined that there was inadequate or insufficient evidence of an association between exposure to 2,4-D, 2,4,5-T or its
470 VETERANS AND AGENT ORANGE: UPDATE 2008 contaminant TCDD, picloram, or cacodylic acid and birth defects in offspring. Additional information available to the committee responsible for Update 1996 led it to conclude that there was limited or suggestive evidence of an association between at least one of the chemicals of interest 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. Later VAO committees have not encountered additional data to merit changing the conclusion that the evidence is inadequate to support an association between exposure to the chemicals of interest 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 neural- tube defects that were reviewed in the current report and in earlier VAO reports can be found in Tables 7-6 and 7-7, respectively. Update of the Epidemiologic Literature Vietnam-Veteran Studies No Vietnam-veteran studies of exposure to the chemicals of interest and birth defects have been published since Update 2006. Occupational Studies One study published since Update 2006 examined occupational exposures and birth defects. Weselak et al. (2008) reported results of the Ontario Farm Fam- ily Health Study. Spouses completed questionnaires that requested the history of pesticide use on the farm. Pregnancies resulting in birth defects were reported by the female study participants. All birth defects were combined for study analyses and exposure was examined by pesticide class, family, and active ingredient for two 3-month periods: before and after conception. After adjustment for infant sex, maternal age at conception, parity, and fever during pregnancy, no association was observed between phenoxy herbicide use and any birth defect during either period. Slightly increased ORs were observed for preconception use of 2,4-D (OR = 1.07, 95% CI 0.55â2.08) and dicamba (OR = 1.67, 95% CI 0.79â3.53). When the outcome was limited to birth defects in male offspring, the asso- ciations increased for both 2,4-D (OR = 1.25, 95% CI 0.56â2.81) and dicamba (OR = 2.42, 95% CI 1.06â5.53). No association with birth defects was observed in analyses that addressed direct use of phenoxy herbicides or 2,4-D specifically by fathers in the 3 months before conception. Two additional articles describing the evaluation of occupational pesticide exposures and birth defects have been published since Update 2006. Lacasana
REPRODUCTIVE EFFECTS AND IMPACTS ON FUTURE GENERATIONS 471 TABLE 7-6â Selected Epidemiologic StudiesâBirth Defects in Offspring of Subjectsa Estimated Exposed Relative Risk Reference Study Population Casesb (95% CI)b VIETNAM VETERANS Studies Reviewed in Update 2002 Kang et al., 2000 Female Vietnam-era veteransâdeployed vs nondeployed (maternal exposure) âLikelyâ birth defects nr 1.7 (1.2â2.2) âModerate-to-severeâ birth defects nr 1.5 (1.1â2.0) Studies Reviewed in Update 2000 AIHW, 1999 Australian Vietnam veteransâvalidation study Cases expected (95% CI) Down syndrome 67 92 expected (73â111) Tracheo-esophageal fistula 10 23 expected (14â32) Anencephaly 13 16 expected (8â24) Cleft lip or palate 94 64 expected (48â80) Absent external body part 22 34 expected (23â45) Extra body part 74 74 expected (nr) Michalek et al., Air Force Ranch Hand veterans 1998a Before service in SEA nr 0.7 (nr) After service in SEA nr 1.5 (nr) Studies Reviewed in Update 1996 Wolfe et al., High-exposure Ranch Hands relative to 1995 comparisons All anomalies 57 1.0 (0.8â1.3) Nervous system 3 nr Eye 3 1.6 (0.4â6.0) Ear, face, neck 5 1.7 (0.6â4.7) Circulatory system, heart 4 0.9 (0.3â2.7) Respiratory system 2 nr Digestive system 5 0.8 (0.3â2.0) Genital system 6 1.2 (0.5â3.0) Urinary system 7 2.1 (0.8â5.4) Musculoskeletal 31 0.9 (0.6â1.2) Skin 3 0.5 (0.2â1.7) Chromosomal anomalies 1 nr Studies Reviewed in VAO AFHS, 1992 Air Force Operation Ranch Hand veteransâ birth defects in conceptions after service in SEA Congenital anomalies 229 1.3 (1.1â1.6) Nervous system 5 1.9 (0.5â7.2) Respiratory system 5 2.6 (0.6â10.7) Circulatory system, heart 19 1.4 (0.7â2.6) continued
472 VETERANS AND AGENT ORANGE: UPDATE 2008 TABLE 7-6â Continued Estimated Exposed Relative Risk Reference Study Population Casesb (95% CI)b Urinary system 21 2.5 (1.3â5.0) Chromosomal 6 1.8 (0.6â6.1) Other 5 2.6 (0.6â10.7) Aschengrau and Vietnam veterans whose children were born at Monson, 1990 Boston Hospital for Women All congenital anomalies (crude OR) vs men without known military service 55 1.3 (0.9â1.9) vs non-Vietnam veterans 55 1.2 (0.8â1.9) One or more major malformations (crude OR) vs men without known military service 18 1.8 (1.0â3.1) vs non-Vietnam veterans 18 1.3 (0.7â2.4) CDC, 1989a Vietnam Experience Studyâinterview data Total anomalies 826 1.3 (1.2â1.4) Nervous system defects 33 2.3 (1.2â4.5) Ear, face, neck defects 37 1.6 (0.9â2.8) Integument 41 2.2 (1.2â4.0) Musculoskeletal defects 426 1.2 (1.1â1.5) Hydrocephalus 11 5.1 (1.1â23.1) Spina bifida 9 1.7 (0.6â5.0) Hypospadias 10 3.1 (0.9â11.3) Multiple defects 71 1.6 (1.1â2.5) Children of veterans reporting high exposure 46 1.7 (1.2â2.4) CDC, 1989b GBDSâhospital records Birth defects 130 1.0 (0.8â1.3) Major birth defects 51 1.2 (0.8â1.9) Digestive system defects 18 2.0 (0.9â4.6) Birth defectsâblack Vietnam veterans only 21 3.4 (1.5â7.6) Donovan et al., Australian Vietnam veterans 1984 Vietnam veterans vs all other men 127 1.0 (0.8â1.3) National Service veteransâVietnam service vs no Vietnam service 69 1.3 (0.9â2.0) Erikson et al., Vietnam veterans identified through CDC 1984a Metropolitan Atlanta Congenital Defects Program Any major birth defects 428 1.0 (0.8â1.1) Multiple birth defects with reported exposure 25 1.1 (0.7â1.7) EOI-5: spina bifida 1 2.7 (1.2â6.2) EOI-5: cleft lip with or without cleft palate 5 2.2 (1.0â4.9) OCCUPATIONAL New Studies Weselak et al., Pregnancies with one or more birth defects in 2008 OFFHS 108 Use on farm, during 3 months before conception, of: Herbicides 24 0.7 (0.4â1.1)
REPRODUCTIVE EFFECTS AND IMPACTS ON FUTURE GENERATIONS 473 TABLE 7-6â Continued Estimated Exposed Relative Risk Reference Study Population Casesb (95% CI)b Male offspring 19 0.9 (0.5â1.6) Direct paternal use 19 0.5 (0.3â1.0) Phenoxy herbicides 12 0.6 (0.3â1.1) Male offspring 9 0.8 (0.4â1.7) Direct paternal use 8 0.4 (0.2â0.9) 2,4-D 10 1.1 (0.6â2.1) Male offspring 7 1.3 (0.6â2.8) Direct paternal use 6 0.6 (0.3â1.5) Dicamba 8 1.7 (0.8â3.5) Male offspring 7 2.4 (1.1â5.5) Use on farm, during 3 months after conception, of: Herbicides 7 0.5 (0.2â1.2) Phenoxy herbicides 9 0.8 (0.4â1.5) 2,4-D 7 1.0 (0.4â2.3) Studies Reviewed in Update 2006 Lawson et al., Wives of workers with measured serum TCDD 2004 in NIOSH cohort 14 nr Studies Reviewed in Update 1998 Kristensen et al., Norwegian farmers (maternal, paternal 1997 exposure) 4,189 1.0 (1.0â1.1) Dimich-Ward Sawmill workers with exposure in upper three et al., 1996 quartiles for any job held up to 3 months before conception Cataracts 11 5.7 (1.4â22.6) Genital organs 105 1.3 (0.9â1.5) Garry et al., 1996 Private pesticide appliers All births with anomalies 125 1.4 (1.2â1.7) Circulatory, respiratory 17 1.7 (1.0â2.8) Gastrointestinal 6 1.7 (0.8â3.8) Urogenital 20 1.7 (1.1â2.6) Musculoskeletal, integumental 30 Maternal age under 30 years 11 0.9 (0.5â1.7) Maternal age over 30 years 19 2.5 (1.6â4.0) Chromosomal 8 1.1 (0.5â2.1) Other 48 Maternal age under 35 years 36 1.1 (0.8â1.6) Maternal age over 35 years 12 3.0 (1.6â5.3) Studies Reviewed in VAO Moses et al., Follow-up of 2,4,5-T male production workers 11 1.3 (0.5â3.4) 1984 Suskind and Follow-up of 2,4,5-T male production workers 18 1.1 (0.5â2.2) Hertzberg, 1984 Smith et al., Follow-up of 2,4,5-T sprayersâsprayers vs 90% CI 1982 non-sprayers 13 1.2 (0.6â2.5) continued
474 VETERANS AND AGENT ORANGE: UPDATE 2008 TABLE 7-6â Continued Estimated Exposed Relative Risk Reference Study Population Casesb (95% CI)b Townsend et al., Follow-up of Dow Chemical plant workers 30 0.9 (0.5â1.4) 1982 ENVIRONMENTAL New Studies Meyer et al., Caseâcontrol study in eastern Arkansas of 2006 hypospadias as function of motherâs residence within 500 m of agricultural pesticide use during gestation weeks 6â16 Dicamba (lb) 0 nr 1.0 > 0â < 0.04 nr 0.5 (0.3â1.0) â¥ 0.04 nr 0.9 (0.4â2.1) Studies Reviewed in Update 2006 Cordier et al., Residents of RhÃ´ne-Alpes region of France 2004 living near municipal solid-waste incinerators (maternal, paternal exposure) Minor anomalies 518 0.9 (0.8â1.1) Chromosomal anomalies 204 1.0 (0.9â1.2) Monogenic anomalies 83 1.1 (0.8â1.4) Unknown or multifactoral etiology 964 1.1 (1.0â1.2) Schreinemachers, Rural or farm residents of Minnesota, Montana, 2003 North Dakota, South Dakota (maternal, paternal exposure) Any birth anomaly 213 1.1 (0.9â1.3) Central nervous system anomalies 12 0.8 (0.5â1.4) Circulatory, respiratory anomalies 39 1.7 (1.1â2.6) Digestive system anomalies 24 0.9 (0.6â1.5) Urogenital anomalies 44 1.0 (0.7â1.5) Musculoskeletal, integumental anomalies 70 1.5 (1.1â2.1) Chromosomal anomalies 17 0.9 (0.6â1.6) Tango, 2004 Investigated multiple pregnancy outcomes in Japan-infant deaths from congenital defects 42 nr, but ns Studies Reviewed in Update 2002 Loffredo et al., Mothers in the BWIS exposed to herbicides 2001 during first trimester (maternal exposure) 8 2.8 (1.2â6.9) Revich et al., Residents of Chapaevsk, Russiaâcongenital 2001 malformations nr nr, but ns ten Tusscher Infants born in Zeeburg, Amsterdam, clinics et al., 2000 1963â1965 with orofacial cleft (maternal exposure) Births in 1963 5 nr, but said to be significant Births in 1964 7 nr, but said to be significant
REPRODUCTIVE EFFECTS AND IMPACTS ON FUTURE GENERATIONS 475 TABLE 7-6â Continued Estimated Exposed Relative Risk Reference Study Population Casesb (95% CI)b Studies Reviewed in Update 2000 GarcÃa et al., Residents of agricultural areas in Spainâat 1998 least median score on chlorophenoxy-herbicide exposure duration (months) index 14 3.1 (0.6â16.9) Studies Reviewed in VAO Fitzgerald et al., Persons exposed to an electric-transformer 1989 fireâtotal birth defects (maternal, paternal exposure) 1 2.1 (0.05â11.85) Mastroiacovo Seveso residents (maternal, paternal, in utero et al., 1988 exposure) 90% CI Zones A, B, Râtotal defects 137 1.0 (0.8â1.1) Zones A and Bâtotal defects 27 1.2 (0.9â1.6) Zones A and Bâmild defects 14 1.4 (0.9â2.2) Stockbauer et al., Persons in Missouri with documented TCDD 1988 soil contamination near residence (maternal, paternal, in utero exposure) Total birth defects 17 0.8 (0.4â1.5) Major defects 15 0.8 (0.4â1.7) Midline defects 4 0.7 (0.2â2.3) Hanify et al., Residents of areas of northland New Zealand 1981 subject to aerial 2,4,5-T spraying birth malformations excluding dislocated All 90% CI or dislocatable hip 164 1.7 (1.4â2.1) All heart malformations 20 3.9 (2.1â7.4) Hypospadias, epispadias 18 5.6 (2.7â11.7) Talipes 52 1.7 (1.2â2.3) Cleft lip 6 0.6 (0.3â1.3) Isolated cleft palate 7 1.4 (0.6â3.2) ABBREVIATIONS: 2,4Â-D, 2,4-dichlorophenoxyacetic acid; 2,4,5-T, 2,4,5-trichlorophenoxyacetic acid; BWIS, BaltimoreâWashington Infant Study; CI, confidence interval; EOI, exposure opportunity index; GBDS, General Birth Defects Study; NIOSH, National Institute for Occupational Safety and Health; nr, not reported; ns, not significant; OFFHS, Ontario Farm Family Health Study; OR, odds ratio; SEA, Southeast Asia; TCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxin. aUnless otherwise indicated, studies show paternal exposure. bGiven when available; results other than estimated risk explained individually. et al. (2006) examined anencephaly and maternal and paternal agricultural work during pregnancy in a caseâcontrol study. However, exposure definition was lim- ited to general job characteristics (such as agricultural work) rather than specific pesticides, so the study did not meet the level of exposure classification required for full review by the committee. Similarly, the work of Carbone et al. (2007) was limited by an exposure definition that considered the probability of exposure to
476 VETERANS AND AGENT ORANGE: UPDATE 2008 TABLE 7-7â Selected Epidemiologic StudiesâNeural-Tube Defects in Offspring of Subjectsa Estimated Exposed Relative Risk Reference Study Population Casesb (95% CI)b VIETNAM VETERANS Studies Reviewed in Update 2000 AIHW, Australian Vietnam veteransâvalidation study Cases expected 1999 (95% CI) Spina bifidaâmaximums 33 expected 50 (22â44) Anencephaly 16 expected 13 (8â24) Studies Reviewed in Update 1996 Wolfe et al., Air Force Operation Ranch Hand personnelâ 1995 neural-tube defects 4c nr Studies Reviewed in VAO CDC, 1989b Vietnam Experience Study Spina bifida Vietnam veteransâ children 9 1.7 (0.6â5.0) Non-Vietnam veteransâ children 5 1.0 Anencephaly Vietnam Veteransâ children 3 nr Non-Vietnam veteransâ children 0 1.0 Erickson CDC birth defects caseâcontrol study et al., Service in Vietnam 1984a,b Spina bifida 19 1.1 (0.6â1.7) Anencephaly 12 0.9 (0.5â1.7) Military records indicate opportunity for exposure Spina bifida 20 2.7 (1.2â6.2) Anencephaly 7 0.7 (0.2â2.8) ADVA, Australian Vietnam veteransâneural-tube defects 16 0.9 (nr) 1983 OCCUPATIONAL Studies Reviewed in Update 1998 Blatter Dutch farmers et al., 1997 Spina bifidaâmoderate, heavy exposure Pesticide use 8 1.7 (0.7â4.0) Herbicide use 7 1.6 (0.6â4.0)d Kristensen Norwegian farmersâspina bifida (maternal, paternal et al., 1997 exposure) Tractor spraying equipment 28 1.6 (0.9â2.7) Tractor spraying equipment, orchards, greenhousese 5 2.8 (1.1â7.1)
REPRODUCTIVE EFFECTS AND IMPACTS ON FUTURE GENERATIONS 477 TABLE 7-7â Continued Estimated Exposed Relative Risk Reference Study Population Casesb (95% CI)b Dimich- Sawmill workers with exposure in upper three Ward et al., quartiles for any job held up to 3 months before 1996 conception Spina bifida, anencephaly 22 2.4 (1.1â5.3) Spina bifida only 18 1.8 (0.8â4.1) Garry et al., Private pesticide appliersâcentral nervous system 1996 defects 6 1.1 (0.5â2.4) ENVIRONMENTAL Studies Reviewed in Update 2006 Cordier Residents of RhÃ´ne-Alpes region of France et al., 2004 (maternal, paternal exposure) 49 0.9 (0.6â1.2) Studies Reviewed in VAO Stockbauer Persons in Missouri with documented TCDD soil et al., 1988 contaminationâcentral nervous system defects (maternal, paternal, in utero exposure) 3 3.0 (0.3â35.9) Hanify Spraying of 2,4,5-T in New Zealand (all exposures) 90% CI et al., 1981 Anencephaly 10 1.4 (0.7â2.9) Spina bifida 13 1.1 (0.6â2.1) ABBREVIATIONS: 2,4,5-T, 2,4,5-trichlorophenoxyacetic acid; CDC, Centers for Disease Control and Prevention; CI, confidence interval; nr, not reported; TCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxin. aUnless otherwise indicated, studies show paternal exposure. bGiven when available; results other than estimated risk explained individually. cOf four neural-tube defects reported in Ranch Hand offspring, two were spina bifida (high dioxin exposure), one spina bifida (low dioxin), one anencephaly (low dioxin); no neural-tube defects reported in comparison cohort; 454 postservice births studied in Ranch Hand veterans; 570 in com- parison cohort. dCalculated from data presented in the paper. eGreenhouse workers would not have been exposed to chemicals of interest. pesticides (all types combined) and was therefore unable to examine the associa- tions related to the specific chemicals of interest for this review. Environmental Studies Meyer et al. (2006) examined maternal residential proximity to agricultural pesticide applications and hypospadias. This population-based study identi- fied cases and controls born in eastern Arkansas in 1998â2002. A geographic- information-system approach was used as a surrogate for pesticide exposure, defined as applications within 500 m of each motherâs residence during gesta- tional weeks 6â16. Land-cover data, providing crop locations and type, and state- wide annual data on crop-specific pesticide use were used to classify maternal
478 VETERANS AND AGENT ORANGE: UPDATE 2008 exposure during pregnancy. Of the 116 pesticides used during the study period, the authors focused on 38 pesticides with existing toxicologic evidence of repro- ductive, developmental, or endocrine-disrupting properties. Of the chemicals of interest in the VAO series, 2,4-D was considered as a possible endocrine disrup- tor that influenced LH and dicamba was tracked as a possible developmental toxicant. Dicamba, but not 2,4-D, was among the 16 pesticides used in proxim- ity to maternal residences. No association with hypospadias was observed with all pesticides combined or with categories based on mode of action and target hormone. Dicamba was among four pesticides to which the cases had signifi- cantly lower average exposure than the controls (p < 0.05). Similarly, with the exception of diclofop-methyl (not a pesticide included as a chemical of interest for VAO committees), no doseâresponse relationships were found in analyses of specific pesticides adjusted for maternal age, race, smoking, and weight gain; timing of first prenatal visit; gestational age; and paternal education. 2,4-D was not examined separately. Two additional articles published since Update 2006 examined environmen- tal exposure to pesticides and birth defects. Felix et al. (2008) defined exposure as herbicide and insecticide use combined, and Clementi et al. (2007) used an ecologic exposure definition (living in an area of high pesticide application). Those broad exposure definitions did not meet the level of exposure specificity required for full review by the committee. Biologic Plausibility Little information is available on the reproductive and developmental effects of exposure to the herbicides discussed in this report. Studies indicate that 2,4-D does not affect male or female fertility and does not produce fetal abnormalities. Offspring of pregnant rodents exposed to 4-(2,4-dichlorophenoxy) butyric acid exhibit a reduced growth rate and increased mortality (Charles et al., 1999) but only after very high doses. Exposure to 2,4-D also alters the concentration and function of reproductive hormones and prostaglandins. One study reported an increased incidence of malformed offspring of male mice exposed to a mixture of 2,4-D and picloram in drinking water; however, paternal toxicity was observed in the high-dose group, and there was no clear doseâresponse relationship (Blakley et al., 1989). Picloram alone produced fetal abnormalities in rabbits at doses that are also toxic to the pregnant animals (John-Greene et al., 1985), but that effect has not been seen in many studies. 2,4,5-T was toxic to fetuses when administered to pregnant rats, mice, and hamsters, but in early studies TCDD contamination confounded attribution of the observed toxicity specifically to the herbicide; its ability to interfere with calcium homeostasis in vitro has been documented and linked to its teratogenic effects on the early development of sea urchin eggs. Cacodylic acid is toxic to rat, mouse, and hamster fetuses at high doses that are also toxic to the pregnant mothers.
REPRODUCTIVE EFFECTS AND IMPACTS ON FUTURE GENERATIONS 479 TCDD is a potent teratogen in all laboratory species that have been studied, but the pattern of induced birth defects is often species-specific. As demonstrated by continuing research, fish, mouse, and avian embryos exhibit substantial altera- tions in craniofacial developmentâshortened jaw in piscine species (Yamauchi et al., 2006), cleft palate in mice (Fujiwara et al., 2008; Jang et al., 2007, 2008; Keller et al., 2007b, 2008; Yamada et al., 2007), and beak malformations in birds (Blankenship et al., 2003). The developing cardiovascular system is also a com- mon target for TCDD-induced teratogenicity (Lahvis et al., 2000; Mehta et al., 2008; Yamauchi et al., 2006). Effects on the fetal kidney (Choi et al., 2006; Keller et al., 2007a,b,c, 2008; Nishimura et al., 2008) and on dental or bone develop- ment (Bursian et al., 2006a,b; Gao et al., 2004, 2007; Ilvesaro et al., 2005; Lind et al., 1999, 2000a,b; Miettinen et al., 2005, 2006; Yasuda et al., 2005) have also been noted frequently after TCDD exposure. The mechanisms by which TCDD induces various birth defects have not been exhaustively determined, but they appear to involve considerable spe- cies specificity and organ specificity. Studies have consistently demonstrated that TCDD-induced developmental toxicity required the AHR. That has been definitively established in mice that lack AHR expression. When pregnant AHR- null mice are exposed to TCDD, the fetuses fail to exhibit any of the typical developmental malformations associated with TCDD exposure. The activated AHR mediates changes in gene transcription, so the inappropriate and sustained activation of AHR by TCDD during development appears to be a key first step in mediating TCDDâs developmental toxicity. Although structural differences in the AHR have been identified among species, it functions similarly in animals and humans. Therefore, a common mechanism mediated by the AHR in which tissue growth and differentiation processes are affected probably underlies the reproductive and developmental toxicity of TCDD in humans and animals. It has been shown that cardiac myocytes and the endothelial lining of the heart and blood vessels are primary target sites of TCDDâs effects on the de- veloping cardiovascular system. CYP1A1 induction or alterations in pathways controlled by vascular endothelial growth factor might mediate the early lesions that result in TCDD-related vascular derangements. That antioxidant treatment provides protection against TCDD-induced embryotoxicity in some systems sug- gests that reactive oxygen species might be involved in the teratogenic effects of exposure to TCDD. Studies in which effects of TCDD on the developing embryo or fetus were investigated after maternal exposure to TCDD; they include an array of animal models and a variety of experimental outcomes. Such laboratory studies have established that maternal exposure to TCDD during pregnancy is associated with a wide variety of birth defects, which depend on the timing of exposure and the species being studied. For instance, Hutt et al. (2008) identified the compaction stage of preimplantation rat embryogenesis as critically sensitive to the effects of TCDD, whereas survival to the blastocyst stage is not compromised.
480 VETERANS AND AGENT ORANGE: UPDATE 2008 Few laboratory studies of potential male-mediated developmental toxicity (and birth defects specifically) attributable to exposure to TCDD and herbicides have been conducted. Feeding of simulated Agent Orange mixtures to male mice produced no adverse effects in offspring; a statistically significant excess of fused sternebrae in the offspring of the two most highly exposed groups was attributed to an anomalously low rate of this defect in the controls (Lamb et al., 1981). It is notable, however, that both the AHR and the ARNT are expressed in the human testis and sperm (Khorram et al., 2004; Shultz et al., 2003), and stud- ies in rodents have shown that TCDD exposure results in significant changes in gene expression in spermatocytes and Sertoli cells; hence, cells of the testis are responsive to TCDD exposure (Kuroda et al., 2005; Yamano et al., 2005). Thus, there is biologic potential for paternal exposure to contribute to TCDD-induced developmental toxicity. Synthesis Embryonic and fetal development is a sensitive toxic outcome of TCDD and DLCs in rodents. It is clear that the fetal rodent is more sensitive to adverse ef- fects of TCDD than the adult rodent; human data are generally lacking, however, and the sensitivity of this outcome in humans is less apparent. Overall, neither of the studies (Meyer et al., 2006; Weselak et al., 2008) considered by the committee provided evidence of an association between the chemicals of interest and birth defects. An association with dicamba exposure and birth defects in male offspring was observed in the study by Weselak et al. (2008), but the interpretation of this finding is limited by the inability to attribute the association with specific birth defects. Given the age of the Vietnam-veteran cohort, publication of additional in- formation on this outcome in the target population of the VAO series is highly unlikely. Conclusions There were no new relevant studies of the association between parental ex- posure to 2,4-D, 2,4,5-T, TCDD, cacodylic acid, or picloram and spina bifida in offspring. The committee concludes that the evidence of an association between exposure to the chemicals of interest and spina bifida is still limited or suggestive. The evidence of an association between exposure to the chemicals of interest and other birth defects is inadequate or insufficient. CHILDHOOD CANCER The American Cancer Society estimated that 10,730 children under 15 years old would have a diagnosis of cancer in the United States in 2008 (ACS, 2008). Treatment and supportive care of children with cancer have improved greatly, and
REPRODUCTIVE EFFECTS AND IMPACTS ON FUTURE GENERATIONS 481 mortality has declined by 49% over the last 30 years. Despite those advances, cancer remains the leading cause of death from disease in children under 15 years old, and 1,490 deaths were projected for 2008 (ACS, 2008). Leukemia is the most common cancer in children. It accounts for about one-third of all childhood cancer cases; leukemia was expected to be diagnosed in nearly 3,540 children in 2008 (ACS, 2008). Of those, nearly 2,000 will have acute lymphocytic leukemia (ALL); most of the rest will have acute myelogenous leukemia. Acute myelogenous leukemia (ICD-9 205) is commonly referred to as acute myeloid leukemia and acute nonlymphocytic leukemia. For consistency, this report uses acute myelogenous leukemia, or simply AML, regardless of us- age in the source materials. ALL is most common in early childhood, peaking at the ages of 2â3 years, and AML is most common during the first 2 years of life. ALL incidence is consistently higher in boys than in girls; AML incidence is similar in boys and girls (NCI, 2001). Through early adulthood, ALL rates are about twice as high in whites as in blacks; AML exhibits no consistent pattern in this respect. Chapter 6 contains additional information on leukemia as part of the discussion of adult cancer. The second-most common group of cancers in children are those of the central nervous systemâthe brain and the spinal cord. Other cancers in children include lymphomas, bone cancers, soft-tissue sarcomas, renal cancers, eye can- cers, and adrenal gland cancers. In contrast with adult cancers, relatively little is known about the etiology of most childhood cancers, especially about potential environmental risk factors and the effect of parental exposures. Conclusions from VAO and Updates The committee responsible for VAO concluded that there was inadequate or insufficient evidence of an association between exposure to 2,4-D, 2,4,5-T, TCDD, picloram, or cacodylic acid and childhood cancers. Additional informa- tion available to the committees responsible for Update 1996 and Update 1998 did not change that conclusion. The committee responsible for Update 2000 reviewed the material in earlier VAO reports and newly available published literature and determined that there was limited or suggestive evidence of an as- sociation between exposure to at least one of the chemicals of interest and AML. After the release of Update 2000, investigators involved in one study discovered an error in their published data. The committee reconvened to evaluate the previ- ously reviewed and new literature regarding AML, and Acute Myelogenous Leu- kemia (IOM, 2002) was produced. It reclassified AML from âlimited/Âsuggestive evidence of an associationâ to âinadequate evidence to determine whether an association exists.â Table 7-8 summarizes the results of the relevant studies. The committees responsible for Update 2000, Update 2002, Update 2004, and Update 2006 reviewed the material in earlier VAO reports and in newly available published literature and agreed that there remained inadequate or insufficient evidence of an association between exposure and childhood cancers.
482 VETERANS AND AGENT ORANGE: UPDATE 2008 TABLE 7-8â Selected Epidemiologic StudiesâChildhood Cancersa Estimated Exposed Relative Risk Reference Study Population Casesb (95% CI)b VIETNAM VETERANS Studies Reviewed in Herbicide/Dioxin Exposure and AML in the Children of Veterans AIHW, Australian Vietnam veteransâ childrenârevised 2001 validation studyâAML 12c 1.3 (0.8â4.0) Studies Reviewed in Update 2000 AIHW, 2000 Australian Vietnam veteransâ childrenâvalidation studyâAML This study, which incorrectly calculated expected number of AML cases, is superseded by AIHW, 2001 above Wen et al., Caseâcontrol study of childrenâs leukemia 2000 AML, ALL Father ever served in Vietnam, Cambodia 117 1.2 (0.9â1.6) < 1 year in Vietnam or Cambodia 61 1.4 (0.9â2.0) > 1 year in Vietnam or Cambodia 49 1.2 (0.8â1.7) AML only Father ever served in Vietnam, Cambodia 40 1.7 (1.0â2.9) < 1 year in Vietnam, Cambodia 13 2.4 (1.1â5.4) > 1 year in Vietnam, Cambodia 16 1.5 (0.7â3.2) Studies Reviewed in VAO CDC, 1989b Vietnam Experience Studyâoutcomes in offspring of veterans Cancer 25 1.5 (0.7â2.8) Leukemia 12 1.6 (0.6â4.0) Field and Cancer in children of Australian Vietnam veterans 4 nr Kerr, 1988 Erikson CDC Birth Defects Studyâchildren of Vietnam et al., 1984b veterans âOtherâ neoplasms 87 1.8 (1.0â3.3) OCCUPATIONAL New Studies Monge Parental occupational exposure to pesticide, et al., 2007 childhood leukemia in Costa Rica Paternal exposure in year before conception to: Herbicides 53 1.2 (0.8â1.7) Phenoxyacetic acids 28 1.0 (0.6â1.6) Picloram (all ALL) 11 1.6 (0.7â3.4) High vs low 8 6.3 (1.0â38.6) Maternal exposure to: Herbicides In year before conception 9 2.0 (0.8â5.0) In 1st trimester 8 5.3 (1.4â20.0) In 2nd trimester 8 5.3 (1.4â20.0) In 3rd trimester 7 2.3 (0.8â6.8) Phenoxyacetic acids in year before conception 4 1.3 (0.4â4.8)
REPRODUCTIVE EFFECTS AND IMPACTS ON FUTURE GENERATIONS 483 TABLE 7-8â Continued Estimated Exposed Relative Risk Reference Study Population Casesb (95% CI)b Studies Reviewed in Update 2006 Chen et al., Parental occupational exposure to pesticide, 2005 childhood GCTs Maternal 32 1.1 (0.7â1.6) Paternal 39 0.9 (0.6â1.3) Reynolds Maternal exposure to agricultural pesticide in et al., 2005b class of âprobable human carcinogensâ (including cacodylic acid) during 9 months before delivery All sites 223 1.0 (0.9â1.2) Leukemias 179 1.2 (0.9â1.5) Central nervous system tumors 31 0.9 (0.5â1.4) Studies Reviewed in Update 2004 Flower Offspring of male pesticide applicators in Iowa from et al., 2004 AHS Maternal exposure to chlorophenoxy herbicides 7 0.7 (0.3â1.5) Paternal exposure to chlorophenoxy herbicides 28 1.3 (0.6â2.6) Maternal exposure to 2,4-D 7 0.7 (0.3â1.6) Paternal exposure to 2,4-D 26 1.3 (0.7â2.4) Studies Reviewed in Update 2000 Heacock Offspring of sawmill workers exposed to fungicides et al., 2000 contaminated with PCDDs, PCDFs Leukemia All workersâ offspringâincidence 11 1.0 (0.5â1.8) Chlorophenate exposure: high- vs low- exposure subjects 5 0.8 (0.2â3.6) Brain cancer All workersâ offspringâincidence 9 1.3 (0.6â2.5) Chlorophenate exposure: high- vs low- exposure subjects 5 1.5 (0.4â6.9) Buckley Childrenâs Cancer Study Groupâexposure to et al., 1989 pesticides, weed killersâAML Any paternal exposure 27 2.3 (p = 0.5) Paternal exposure over 1,000 days 17 2.7 (1.0â7.0) Maternal exposure over 1,000 days 7 undefined ENVIRONMENTAL New Studies Cooney Caseâcontrol study of Wilmsâ tumor in United States et al., 2007 and Canada Maternal report of household use of herbicides from month before conception through childâs diagnosis 112 1.0 (0.7â1.4) Rudant Caseâcontrol study of childhood hematopoietic et al., 2007 malignancies in France Maternal household herbicide use during pregnancy continued
484 VETERANS AND AGENT ORANGE: UPDATE 2008 TABLE 7-8â Continued Estimated Exposed Relative Risk Reference Study Population Casesb (95% CI)b Acute leukemia 53 1.5 (1.0â2.2) Without paternal exposure 4 5.0 (1.3â19.0) All ALL nr 1.7 (1.2â2.5) Common B-cell ALL nr 1.9 (1.3â2.9) Mature B-cell ALL nr 1.5 (0.3â6.4) T-cell ALL nr 0.5 (0.1â2.0) AML nr 1.2 (0.5â2.8) HD 9 1.1 (0.5â2.4) Without paternal exposure 0 nr Nodular sclerosis nr 1.3 (0.5â3.1) Mixed cell nr 0.8 (0.1â6.6) NHL 14 1.5 (0.8â2.7) Without paternal exposure 0 nr Burkittâs lymphoma nr 1.7 (0.7â4.0) B-cell lymphoblastic nr 0.7 (0.2â3.0) T-cell lymphoblastic nr 2.6 (0.7â9.0) Anaplastic large cell nr 1.4 (0.3â2.8) Studies Reviewed in Update 2006 Chen et al., Childhood GCTs residential exposure to herbicides 6 2006 months before conception, during gestation, through breastfeeding period Maternal exposure 47 1.3 (0.9â1.7) Daughters 36 1.4 (1.0â2.0) Sons 11 1.0 (0.5â1.8) Paternal exposure 90 1.0 (0.7â1.3) Daughters 32 1.2 (0.7â2.0) Sons 58 1.0 (0.7â1.4) Studies Reviewed in Update 2002 Daniels Neuroblastoma risk in children (caseâcontrol study) et al., 2001 (as reported by both parents) Pesticides in home (used ever) nr 1.6 (1.0â2.3) Herbicides in garden nr 1.9 (1.1â3.2) Pesticides in garden nr 2.2 (1.3â3.6) Kerr et al., Neuroblastoma risk in children 2000 Maternal occupational exposure to insecticides 40 2.3 (1.4â3.7) Paternal exposure to dioxin 7 6.9 (1.3â68.4) Studies Reviewed in Herbicide/Dioxin Exposure and AML in the Children of Veterans Kristensen Children of agricultural workers in Norway et al., 1996 Children with AML whose parents purchased pesticides 12 1.4 (0.6â2.9) Studies Reviewed in Update 2000 Meinert Childhood cancerâpopulation-based caseâcontrol et al., 2000 study Leukemia Paternal exposure year before pregnancy 62 1.5 (1.1â2.2) Paternal exposure during pregnancy 57 1.6 (1.1â2.3)
REPRODUCTIVE EFFECTS AND IMPACTS ON FUTURE GENERATIONS 485 TABLE 7-8â Continued Estimated Exposed Relative Risk Reference Study Population Casesb (95% CI)b Maternal exposure year before pregnancy 19 2.1 (1.1â4.2) Maternal exposure during pregnancy 15 3.6 (1.5â8.8) Lymphomas Paternal exposure year before pregnancy 11 1.5 (0.7â3.1) Paternal exposure during pregnancy 10 1.6 (0.7â3.6) Maternal exposure year before pregnancy 3 2.9 (0.7â13) Maternal exposure during pregnancy 4 11.8 (2.2â64) Pearce and Renal cancer in subjects (1â15 years old) with Parker, 2000 paternal occupation in agriculture 21 0.9 (0.2â3.8) Infante- Childhood ALL in households using herbicidesâ Rivard population-based caseâcontrol study et al., 1999 Exposure during pregnancy 118 1.8 (1.3â2.6) Exposure during childhood 178 1.4 (1.1â1.9) Studies Reviewed in Update 1996 Pesatori Seveso residents 0â19 years oldâ10-year follow-up, et al., 1993 morbidity, all exposure zones All cancers 17 1.2 (0.7â2.1) Ovary, uterine adnexa 2 nr (0 cases expected) Brain 3 1.1 (0.3â4.1) Thyroid 2 4.6 (0.6â32.7) HD 3 2.0 (0.5â7.6) Lymphatic leukemia 2 1.3 (0.3â6.2) Myeloid leukemia 3 2.7 (0.7â11.4) Bertazzi Seveso residents 0â19 years oldâ10-year follow-up, et al., 1992 mortality, all exposure zones All cancers 10 7.9 (3.8â13.6) Leukemias 5 3.9 (1.2â1.8) Lymphatic leukemia 2 1.6 (0.1â4.5) Myeloid leukemia 1 0.8 (0.0â3.1) Leukemia, others 2 1.6 (0.1â4.6) Central nervous system tumors 2 1.6 (0.1â4.6) ABBREVIATIONS: 2,4Â-D, 2,4-dichlorophenoxyacetic acid; AHS, Agricultural Health Study; AIHW, Australian Institute for Health and Welfare; ALL, acute lymphocytic leukemia; AML, acute myeloid leukemia; CDC, Centers for Disease Control and Prevention; CI, confidence interval; GCT, germ-cell tumor; HD, Hodgkinâs disease; NHL, non-Hodgkinâs lymphoma; nr, not reported; PCDD, polychlo- rinated dibenzodioxin; PCDF, polychlorinated dibenzofuran. aUnless otherwise indicated, studies show paternal exposure. bGiven when available; results other than estimated risk explained individually. cOf the 12, nine were observed, three additional cases estimated to have occurred in portion of cohort whose data were not validated.
486 VETERANS AND AGENT ORANGE: UPDATE 2008 Update of Epidemiologic Literature Vietnam-Veteran Studies No Vietnam-veteran studies of exposure to the chemicals of interest and childhood cancer have been published since Update 2006. Occupational Studies One paper (Monge et al., 2007) addressed occupational exposure to pesti- cides and the risk of childhood leukemia in Costa Rica. All cases of leukemia in infants and children (up to 14 years old at diagnosis) in Costa Rica in 1995â2000 were identified at the Cancer Registry and at Childrenâs Hospital of Costa Rica. Population-based controls were identified from the National Birth Registry by computerized randomization and were frequency-matched by year of birth. Data were collected via interviews with both parents of the 300 case and 579 control children. Parents who worked in agriculture or livestock production completed an additional interview. Pesticide use and agricultural tasks were ascertained from 12 months before conception until the diagnosis of cancer for the cases and the interview date for the controls. Exposures to any herbicides were assessed during all periods of interest to the researchers (the year before conception, the three trimesters of pregnancy, and the first year of life), but only paternal exposures before conception and maternal exposure before and during gestation could be of any possible relevance to the circumstances under which Vietnam veterans were exposed. Paternal exposure to herbicides during the year before conception was slightly associated with all types of leukemia (OR = 1.2, 95% CI 0.8â1.7). Picloram was one of only five pesticides to which the parents of more than three cases had recorded exposure, and all 11 cases of leukemia involving paternal exposure to picloram were specifically ALL; the association with any paternal exposure to picloram during the year before conception was increased (OR = 1.6, 95% CI 0.7â3.4). The association was greater, but of borderline significance, after high exposure than after low exposure (OR = 6.3, 95% CI 1.0â38.6). Paternal exposure to phenoxyacetic acids was not associated with total leukemia (OR = 1.0, 95% CI 0.6â1.6). Overall, the ORs were higher, but less precise, for maternal exposure to all herbicides combined during the defined periods. The OR for maternal exposure to herbicides before conception and total leukemia was 2.0 (95% CI 0.8â5.0). An increased, but imprecise, risk was observed for maternal exposure to herbi- cides during the first two trimesters of pregnancy (OR = 5.3, 95% CI 1.4â20.0). Maternal exposure to phenoxyacetic acids, with four exposed cases, was weakly associated with total leukemia (OR = 1.3, 95% CI 0.4â4.8). An additional study published since Update 2006 examined paternal occupa- tional exposure to âpesticides or herbicidesâ and childhood cancer (Pearce et al., 2006). However, the exposure definition for analysis was limited to job titles that
REPRODUCTIVE EFFECTS AND IMPACTS ON FUTURE GENERATIONS 487 were likely to include exposure to pesticides and therefore did not meet the level of exposure specificity required for full review by the committee. Environmental Studies Cooney et al. (2007) examined household pesticide use and Wilmsâ tumor in a population-based caseâcontrol study. Cases, all registered with the National Wilmsâ Tumor Study, were patients under 16 years old who had Wilmsâ tumor newly diagnosed in the United States or Canada in 1999â2002. After the exclu- sion of 512 cases lacking physician or hospital approval for participation, 653 eli- gible cases remained, and 523 case mothers completed interviews. Random-digit dialing (RDD) was used to identify 682 eligible controls, who were frequency- matched on age at diagnosis and geographic residence. Of the control mothers, 517 completed interviews. Structured computer-assisted telephone interviews were used to gather information from mothers of cases and controls, including in- formation on use of household pesticides from the month before pregnancy to the date of the childâs diagnosis (or referent date); only the month before conception is relevant for the exposures being evaluated in this report, but exposures would be dominated by the period of gestation and childhood. No association with that broader definition of herbicide exposure was observed in the study (OR = 1.0, 95% CI 0.7â1.4). Rudant et al. (2007) also examined household pesticide use in relation to the risk of childhood hematopoietic cancers in the ESCALE study, a French national population-based caseâcontrol study designed to examine infectious, environmental, and genetic factors in childhood cancers (leukemia, lymphoma, neuroblastoma, and brain tumor). Cases less than 15 years old who received new diagnoses during the 2003â2004 study period were identified from each of the pediatric-oncology departments with support of the French National Registry of Childhood Blood Malignancies. The population-based controls were randomly selected and frequency-matched on age and sex. Subjects who were adopted or whose mother had died, had a psychiatric problem, or did not speak French were excluded. Structured telephone interviews were used to obtain data from the mothers of cases and controls and included questions about household pesticide use by the mothers during pregnancy. Pesticides were classified by functional groups (such as herbicides). Maternal exposure to herbicides (all types combined) during pregnancy was associated with acute leukemia (AL) (OR = 1.5, 95% CI 1.0â2.2) and non-Hodgkinâs lymphoma (NHL) (OR = 1.5, 95% CI 0.8â2.7); no association was observed with Hodgkinâs disease (HD) (OR = 1.1, 95% CI 0.5â2.4). The studyâs assessment of postconception paternal exposure, which was considerably more common than maternal exposure, is not relevant for the present review. However, the pattern of associations with paternal exposure was similar to the pattern of associations between maternal exposure and AL (OR = 1.2, 95% CI 1.0â1.4), NHL (OR = 1.5, 95% CI 1.0â2.2), and HD (OR = 1.3, 95% CI 0.8â2.2). When the definition of exposure was limited to maternal exposure,
488 VETERANS AND AGENT ORANGE: UPDATE 2008 the number of exposed AL cases decreased to four, but the observed association with AL increased (OR = 5.0, 95% CI 1.3â19.0). No NHL or HD cases were considered to have had only maternal exposure. When malignancy subtypes were examined, maternal household use of herbicides was associated with all types of ALL (OR = 1.7, 95% CI 1.2â2.5) and with the common B-cell subtype of ALL (OR = 1.9, 95% CI 1.3â2.9). The Centers for Disease Control and Prevention investigated environmental factors that might influence childhood leukemia in Churchill County, Nevada, where a higher than expected number of cases were diagnosed in 1997â2002 (Rubin et al., 2007). This cross-sectional case-comparison study enrolled 14 of the 15 eligible families of children who had leukemia. RDD was used to identify the 55 participating comparison families (child and caretaking adults living in the home), which were matched on case childrenâs sex and years of birth. Biologic and environmental samples were collected from each of the families, and family members were interviewed. None of the 31 nonpersistent pesticides was associ- ated with leukemia, although geometric mean urinary concentrations of 2,4-D and 2,4,5-T in the entire study sample were above the reference (National Health and Nutrition Examination Survey) geometric mean. No association between the 11 persistent pesticides measured in serum and cases status was observed. Two additional studiesâby Carozza et al. (2008) and Walker et al. (2007)â examined environmental exposure to pesticides and childhood cancer. They were limited by definition of exposure on the basis of residence in counties of greater agricultural activity and did not meet the level of exposure specificity required for full review by the committee. Biologic Plausibility Paternal or maternal exposure to xenobiotics potentially could increase the susceptibility of offspring to cancer through multiple mechanisms. Susceptibility could be increased by inheriting a genetic predisposition, which by itself could increase the development of cancer or the likelihood of developing cancer after future exposure to a carcinogen; the mother or father would transmit either an acquired genetic defect or an epigenetic alteration that predisposed the child to cancer. Alternatively, a maternally mediated increase in susceptibility to child- hood cancer could result from direct exposure of a child in utero or via lactation to a xenobiotic that induces epigenetic alterations that increase cancer susceptibil- ity or is itself carcinogic. It has been shown that prenatal TCDD exposure of rats is associated with al- tered mammary gland differentiation and an increase in the number of mammary adenocarcinomas (Brown et al., 1998). A recent studyâs demonstration that early postnatal TCDD exposure does not increase mammary-cancer risk (Desaulniers et al., 2004) is consistent with the finding that TCDD-induced changes in utero mediate the increase in cancer susceptibility (Fenton et al., 2000, 2002). Devel- opmental epigenetic alterations may be involved in those prenatal effects. TCDD
REPRODUCTIVE EFFECTS AND IMPACTS ON FUTURE GENERATIONS 489 has been shown to suppress the expression of two tumor-suppressor genes, p16Ink4a and p53, via an epigenetic mechanism that appears to involve DNA methylation (Ray and Swanson, 2004). Similarly, it was reported that prenatal TCDD exposure increases methylation of two growth-related imprinted genes, H19 and Igf2, in the developing fetus (Wu et al., 2004). Although there is no direct evidence from animal models that TCDD in- creases the risk of childhood cancers, such as acute leukemia or germ-cell tumors, emerging research suggests that prenatal TCDD exposure can disrupt epigenetic imprinting patterns and alter organ differentiation, which could contribute to an increased susceptibility to cancer later in life. A recent study has shown that chromosomal rearrangements associated with childhood ALL are evident in the neonatal blood spots; this suggests that childhood leukemias begin before birth and that maternal and perinatal exposures to xenobiotics may contribute to ge- netic mutations (Smith et al., 2005). Synthesis Two of the four studies reviewed by the committee report increased ORs with respect to herbicide exposure and childhood cancer. Monge et al. (2007) found that paternal exposure to picloram was associated with ALL, but the finding was limited by the small number of exposed cases, which resulted in imprecise esti- mates of risk; no association between paternal exposure to phenoxyacetic acids and leukemia was observed. Maternal exposures in the same study showed an increased risk of total leukemia, but the maternal exposure definition was limited to all herbicides combined. Analyses by pesticide subtypes found no association with maternal exposure to phenoxyacetic acids. Similarly, Rudant et al. (2007) reported an increased OR for maternal exposure to herbicides during pregnancy and ALL. The interpretation of those findings for the purposes of this review is limited by the nonspecific nature of the definition of exposure to include all herbicides. Conclusions On the basis of the evidence reviewed here and in previous VAO reports, the committee concludes that there is inadequate or insufficient evidence of an as- sociation between exposure to the chemicals of interest and childhood cancers. EFFECTS OCCURRING LATER IN OFFSPRINGâS LIFE OR IN LATER GENERATIONS In response to a special request from the Department of Veterans Affairs, continuing inquiries from the veterans themselves and their families, and some- what more attention in research efforts, this update addresses whether it is now necessary and feasible to assess associations between exposure to the herbi-
490 VETERANS AND AGENT ORANGE: UPDATE 2008 cides sprayed in Vietnam and health effects in the children and grandchildren of Vietnam veterans that have not been formally reviewed in the VAO series. The additional outcomes may include effects (other than cancer) in the children that become apparent after the first year of life and that may be related to maternal or paternal exposures (for example, cognitive and behavioral outcomes and effects on sexual development). In addition, the committee explored the possibility of transgenerational effects resulting from exposure-related epigenetic changes, either in the parents or fetuses, that would result in adverse health effects in later generations (such as grandchildren). Problems Detected in Children After First Year of Life There is growing evidence from laboratory animal and human studies that exposures during fetal development can lead to adverse effects later in life that are not immediately apparent as structural malformations or functional deficits. Several reports of studies in animals and exposed humans suggest that prenatal exposure to TCDD or to DLCs can impair brain development and induce neu- robehavioral deficits. Outcomes can be subtle, ranging from altered learning and memory to modification of sex-related behavior. The mechanisms of those ef- fects are unclear. Animal studies have shown that perinatal TCDD exposure can decrease neuron number (Hojo et al., 2006), modify gene expression in various parts of the brain (Chang SF et al., 2005; Fujita et al., 2006; Mitsui et al., 2006; Nayyar et al., 2003), and reduce or reverse sexual dimorphic brain development (Chang SF et al., 2005; Ikeda et al., 2005b). Such changes can be associated with altered behavior and learning in rodents (Ikeda et al., 2005b; Mitsui et al., 2006) and primates (Negishi et al., 2006). Human studies have found correlations between maternal, placental, and breast-milk concentrations of dioxins and PCBs and childrenâs behavior in sev- eral hundred Dutch motherâinfant pairs since 1990 (Koopman-Essenboom et al., 1994; Vreugdenhil and Weisglas-Kuperus, 2000; Vreugdenhil et al., 2002). A study of human exposure to background concentrations of dioxins, furans, and PCBs during prenatal development (Nakajima et al., 2006) suggested a greater association between exposure and reduced motor development in 6-month-old infants than between exposure and mental development; however, few significant correlations among dozens of comparisons with specific congeners that have low relative potency (TEFs). Animal studies have repeatedly shown adverse effects of prenatal TCDD exposure on various reproductive characteristics of the offspring. Studies in rodents have shown that a single maternal dose of TCDD produces malforma- tions of the external genitalia and functional reproductive alterations in female progeny, including decreased fertility rate, reduced fecundity, cystic endometrial hyperplasia, and disrupted estrous cycles. Those effects depend on the timing of exposure. In agreement with earlier work (Dienhart et al., 2000; Gray et al.,
REPRODUCTIVE EFFECTS AND IMPACTS ON FUTURE GENERATIONS 491 1997b), recent research (Kakeyama et al, 2008) found that prenatal exposure to a low dose of TCDD affected the onset of puberty in the female offspring of rats. Several reports (Gray et al., 1997a; Mably et al., 1992; Ohsako et al., 2002) have indicated that development of the male reproductive system is exceptionally sensitive to in utero and lactational TCDD exposure. Maternal exposure to TCDD impairs prostate growth and seminal vesicle weight and branching and decreases sperm production and caudal epididymal sperm number in offspring. Work on the differentiation of mammary tissue in rats (Brown et al., 1998; Fenton et al., 2000, 2002) has demonstrated that TCDD produces changes in utero that increase susceptibility to cancer in adulthood. Similar effects on the reproductive characteristics of the offspring have been reported in humans. Reviewers (Damgaard et al., 2002; Landrigan et al., 2003; Teilmann et al., 2002; Wikiera et al., 2007) have hypothesized that the world- wide reports of precocious puberty, reduced semen quality, and other forms of reproductive dysfunction are related to exposure from before birth to endocrine disruptors, including dioxins, at environmental concentrations. Demonstration of precocious puberty in the daughters of women accidentally exposed to poly- brominated biphenyls (Blanck et al., 2000) suggested the plausibility of such activity of DLCs. Human Effects in Later Generations As the cohort of Vietnam veterans ages, the veteransâchildren are well into their reproductive years, and concerns have been expressed as to whether her- bicide exposures experienced in Vietnam might be having adverse effects on another generation. Growing awareness of the possibility of epigenetic changes (that is, heritable changes in genome function that occur without a change in primary DNA sequences) arising from exposure to the chemicals of interest now increases the plausibility of such transgenerational findings (Cordier, 2008), whereas in previous review cycles VAO committees had been confident that di- oxin (the primary suspect) would not directly produce heritable genetic damage. Epidemiologic investigation of this possibility will be even more challenging to conduct than research on adverse effects on the first generation, but recently recognized epigenetic mechanisms that are the focus of intensive research could consititute a mechanism by which such outcomes might occur. Lasting gene expression changes responsible for the phenotypic changes associated with a disease state are the result of stable alterations of the genetic material by either genetic or epigenetic mechanisms. Several factorsâsuch as chemotherapy, mutagenic agents, endocrine disruptors, and environmental tox- insâalter DNA by one or the other mechanism (Anway et al., 2005; Barber et al., 2002). In mammalian development, long-lasting changes of gene expression result from alterations in DNA methylation patterns, the most common form of epigenetic change modification of the genome (Morgan et al., 2005). DNA meth-
492 VETERANS AND AGENT ORANGE: UPDATE 2008 ylation is associated with permanent changes in the genome (Baylin and Chen, 2005) and is thought to be a strong mechanism for modifying gene expression and to have an important role in normal development and in maintenance of ge- nome stability (Jaenisch and Bird, 2003). Several diseases result from epigenetic imprinting malformations (Jiang YH et al., 2004), including some forms of cancer associated with DNA methylation aberrations (Roundtree et al., 2001). Current understanding is that epigenetic mechanisms are responsible for phenotypic changes induced by various chemicals and agents that act primarily through DNA damage, such as heavy metals, some base analogues, ionizing radiation, reactive oxygen species, and smoke components. Those and other stress-inducing factors, such as pesticides and some hormones (for example, estradiol), may alter DNA methylation states or histone acetylation states. A growing body of evidence suggests that epigenetic factors play a critical role in the development of cancer (Nagasaka et al., 2008). The best-understood epigenetic alteration is DNA methylation that occurs in cytosine residues that form part of CpG islands, so called for their higher content of CpG dinucleotides. CpG dinucleotides occur in gene-promoter regions at fre- quencies greater than would be expected on the basis of random distribution. Ad- dition of a methyl group to the 5â²-carbon of the cytosine in a CpG dinucleotide, if it occurs at multiple cytosines in a particular CpG island, is often associated with silencing of the gene by recruitment of methyl-binding proteins that create a chro- matin conformation that leads to gene repression. During cancer development, epigenetic silencing by promoter hypermethylation is one of the mechanisms that inactivate tumor-suppressor genes. Unlike promoters of tumor-suppressor genes, other DNA sequences of the genome are normally methylated, and their meth- ylation is an important part of normal development, cellular differentiation, and X-chromosome inactivation; at an extreme, changes in their methylation status may lead to specific human disease states (Ehrlich, 2003). Methylation status is genetically determined, but recent work in animal model systems suggests that environmental exposures, particularly during fetal development and early life, also induce epigenetic changes in somatic tissues (Cutfield et al., 2007) that can be passed through multiple generations, although they do not cause DNA muta- tions (Anway and Skinner, 2008). It is during embryonic development that the organism is most sensitive to injury by chemicals and environmental toxins (Yamazaki et al., 2003). That is when lineage-specific and germ-lineâspecific DNA-methylation patterns are established (Reik and Walter, 2001). The lineage-specific pattern establishes the DNA-methylation pattern for the various somatic cells after fertilization. The germ-lineâspecific pattern is established during gonadal sex determination (Reik et al., 2001) and is critical for gene imprinting (Lucifero et al., 2004). Unlike lineage-specific DNA-methylation programming, changes in germ-lineâspecific programming can alter heritable DNA-methylation profiles and result in a trans- generational phenotype. Alterations in lineage-specific DNA methylation po-
REPRODUCTIVE EFFECTS AND IMPACTS ON FUTURE GENERATIONS 493 tentially lead to developmental defects and, in the worst case, embryonic death (Reik et al., 2001). Because of its status as a persistent environmental contaminant of great concern and its known estrogenic activity, dioxin is often mentioned as a chemi- cal that might be expected to produce adverse effects via epigenetic mechanisms (Edwards and Myers, 2007). Research into dioxinâs potential as an epigenetic agent is in its early stages, but there have been several reports that dioxin has such properties. For instance, Wu et al. (2004) have demonstrated that TCDD exposure of mouse embryos before implantation in unexposed females resulted in reduced fetal weight in association with reduced expression and increased methylation of imprinted genes. Another mode of epigenetic change is modification of the spatial arrangement of chromosomes, which correlates with gene expression and with how cells differentiate, and Oikawa et al. (2008) have found that TCDD, through the AHR, modifies the position of chromosomes in the interphase nuclei of human pre-adipocytes. Failure to comprehend how something might happen is not a scientifically legitimate reason for not inquiring into whether it has occurred. Nonetheless, de- spite the profound concern of veterans and their families that herbicide exposures in Vietnam were having adverse effects on the health of their children and now grandchildren, the longstanding biologic conviction that there was no plausible mechanism by which non-genotoxic agents like the chemicals of concern in these VAO reports could produce paternally-mediated transgenerational effects could only discourage undertaking the arduous work of conducting epidemiologic studies on such health problems in older offspring and the second generation. The developing understanding of epigenetic mechanisms leads the committee to conclude that it is considerably more plausible than previously believed that ex- posure to the herbicides sprayed in Vietnam might have caused transgenerational effects. Such potential would most likely be attributable to the TCDD contami- nant in Agent Orange. Consequently, this committee recommends that toxicologic research be conducted to address and characterize TCDDâs potential for inducing epigenetic modifications and is more convinced that additional epidemiologic study would be a worthwhile investment of resources. Efficient conduct of epidemiologic investigations into whether such effects are being manifested in the grandchildren of Vietnam veterans will probably require the development of some innovative techniques and protocols. The de- sign of such studies poses additional challenges compared with the traditional caseâcohort or cohort studies in that large numbers of subjects need to be tracked in multiple generations, exposures need to be reconstructed as specifically as pos- sible for critical periods in each individualâs life, and the effects to be appraised could be quite diverse. Although there could be problems of selection bias, start- ing with previously defined cohorts of deployed and nondeployed Vietnam-era veterans would probably be the most efficient way to address the question of whether the incidence of health problems is increased in the mature children
494 VETERANS AND AGENT ORANGE: UPDATE 2008 and grandchildren of Vietnam veterans. Only a single temporal pattern of expo- sureâpreconception exposure of fathers (almost exclusively)âwould need to be contrasted with a cleanly defined absence of such preconception exposure, so the hypothesis could be tested more precisely than in instances in which paternal or maternal exposure could occur before conception, during gestation, and after birth. It can be expected that it is not going to be possible so many decades after potential exposure to herbicides in Vietnam to determine original exposures ac- curately, so it would be advantageous to continue work with study populations whose serum TCDD has been measured. SUMMARY Synthesis The studies reviewed for this update did not find any significant associa- tions between the relevant exposures and reproductive outcomes. The scientific evidence supports the biologic plausibility of a connection between exposure to the chemicals of interest and reproductive effects, but the epidemiologic studies of occupational cohorts, exposed communities, and Vietnam veterans have not provided conclusive evidence of any additional associations between exposures and an array of reproductive outcomes and conditions in the offspring of exposed parents. The mechanisms by which the chemicals exert their biologic effects are still subjects of scientific investigation. With the aging of the Vietnam-veteran population, additional studies of fertility, spontaneous abortion, and sex ratio cannot be expected, although there may be additional studies of reproductive outcomes in other populations after exposure to the chemicals of interest. The possibility that structural or functional abnormalities will be manifested in the maturing offspring of exposed people will continue to be of interest. In addi- tion, the committee strongly recommends that careful consideration be given to systematically evaluating whether recently recognized mechanisms of epigenetic modification mean that there could be long-term consequences of herbicide expo- sure for the health of the progeny of Vietnam veterans into later generations. Conclusions There is inadequate or insufficient evidence of an association between ex- posure to 2,4-D, 2,4,5-T, TCDD, picloram, or cacodylic acid and endometriosis; semen quality; infertility; spontaneous abortion; late fetal, neonatal, or infant death; low birth weight or preterm delivery; birth defects other than spina bifida; and childhood cancers. There is limited or suggestive evidence of an association between exposure to the chemicals of interest and spina bifida. There is some evidence of altered hormone concentrations, but the degree to which testosterone concentration may be modified is not great enough for clinical consequences to be expected.
REPRODUCTIVE EFFECTS AND IMPACTS ON FUTURE GENERATIONS 495 There is limited or suggestive evidence that the specific combination of pa- ternal exposure to TCDD is not associated with risk of spontaneous abortion. REFERENCES ACS (American Cancer Society). 2008. Cancer Facts and Figures 2008. http://www.cancer.org/Â downloads/STT/2008CAFFfinalsecured.pdf (Accessed March 30). ADVA (Australia Department of Veterans Affairs). 1983. CaseâControl Study of Congenital Anoma- lies and Vietnam Service. Canberra, Australia. AFHS (Air Force Health Study). 1992. An Epidemiologic Investigation of Health Effects in Air Force Personnel Following Exposure to Herbicides. Reproductive Outcomes. Brooks AFB, TX: USAF School of Aerospace Medicine. AL-TR-1992-0090. 602 pp. AIHW (Australian Institute of Health and Welfare). 1999. Morbidity of Vietnam Veterans: A Study of the Health of Australiaâs Vietnam Veteran Community: Volume 3: Validation Study. Canberra, Australia. AIHW. 2000. Morbidity of Vietnam veterans. Adrenal gland cancer, leukaemia and non-Hodgkinâs 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-Hodgkinâs lymphoma: Supplementary report no. 2. Revised edition (AIHW cat. No. PHE 34). Canberra, Australia: AIHW. Alberman E. 1984. Low birth weight. In: Bracken MB, ed. Perinatal Epidemiology. New York: Oxford University Press. Pp. 86â98. Alexander GR, Slay M. 2002. Prematurity at birth: Trends, racial disparities, and epidemiology. Men- tal Retardation and Developmental Disabilities Research Reviews 8(4):215â220. Alexander GR, Kogan M, Bader D, Carlo W, Allen M, Mor J. 2003. US birth weight/gestational age-specific neonatal mortality: 1995â1997 rates for whites, Hispanics, and blacks. Pediatrics 111(1):e61âe66. Anway MD, Skinner MK. 2008. Epigenetic programming of the germ line: Effects of endocrine disruptors on the development of transgenerational disease. Reproductive and Biomedical Online 16:23â25. Anway MD, Cupp AS, Uzumcu M, Skinner MK. 2005. Epigenetic transgenerational actions of en- docrine disruptors and male fertility. Science 308:1466â1469. Arbuckle TE, Lin Z, Mery LS. 2001. An exploratory analysis of the effect of pesticide exposure on the risk of spontaneous abortion in an Ontario farm population. Environmental Health Perspec- tives 109(8):851â857. Aschengrau A, Monson RR. 1989. Paternal military service in Vietnam and risk of spontaneous abor- tion. Journal of Occupational Medicine 31(7):618â623. 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):1218â1224. Axmon A, Rylander L, StrÃ¶mberg U, Hagmar L. 2000. Miscarriages and stillbirths in women with a high intake of fish contaminated with persistent organochlorine compounds. International Archives of Occupational and Environmental Health 73(3):204â208. Baccarelli A, Giacomini SM, Corbetta C, Landi MT, Bonzini M, Consonni D, Grillo P, Patterson Jr DG, Pesatori AC, Bertazzi PA. 2008. Neonatal thyroid function in Seveso 25 years after mater- nal exposure to dioxin. PLoS Medicine 5(7):1133â1142. â Throughout the report the same alphabetic indicator following year of publication is used con- sistently for the same article when there were multiple citations by the same first author in a given year. The convention of assigning the alphabetic indicator in order of citation in a given chapter is not followed.
496 VETERANS AND AGENT ORANGE: UPDATE 2008 Barber R, Plumb MA, Boulton E, Roux I, Dubrova YE. 2002. Elevated mutation rates in the germ line of first- and second-generation offspring of irradiated male mice. Proceeding of the National Academy of Sciences of the United States of America 99:6877â6882. Barnett KR, Tomic D, Gupta RK, Babus JK, Roby KF, Terranova PF, Flaws JA. 2007a. The aryl hydrocarbon receptor is required for normal gonadotropin responsiveness in the mouse ovary. Toxicology and Applied Pharmacology 223(1):66â72. Barnett KR, Tomic D, Gupta RK, Miller KP, Meachum S, Paulose T, Flaws JA. 2007b. The aryl hydrocarbon receptor affects mouse ovarian follicle growth via mechanisms involving estradiol regulation and responsiveness. Biology of Reproduction 76(6):1062â1070. Baylin SB, Chen WY. 2005. Aberrant gene silencing in tumor progression: Implications for control of cancer. Cold Spring Harbor Symposia on Quantitative Biology 70:427â433. Bell DR, Clode S, Fan MQ, Fernandes A, Foster PM, Jiang T, Loizou G, MacNicoll A, Miller BG, Rose M, Tran L, White S. 2007b. Toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin in the develop- ing male Wistar(Han) rat. I: No decrease in epididymal sperm count after a single acute dose. Toxicological Sciences 99(1):214â223. Berkowitz GS, Papiernik E. 1993. Epidemiology of preterm delivery. Epidemiologic Reviews 15: 414â443. 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):118â123. Blakley PM, Kim JS, Firneisz GD. 1989. Effects of paternal subacute exposure to Tordon 202c on fetal growth and development in CD-1 mice. Teratology 39(3):237â241. Blanck H, Marcus M, Tolbert PE, Rubin C, Henderson A, Hertzberg V, Zhang R, Cameron L. 2000. Age at menarche and tanner stage in girls exposed in utero and postnatally to polybrominated biphenyl. Epidemiology 11(6):642â647. Blankenship AL, Hilscherova K, Nie M, Coady KK, Villalobos SA, Kannan K, Powell DC, Bursian SJ, Giesy JP. 2003. Mechanisms of TCDD-induced abnormalities and embryo lethality in white leghorn chickens. Comparative Biochemistry and Physiology. Toxicology and Pharmacology 136(1):47â62. 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):283â291. Bloom AD, ed. 1981. Guidelines for Studies of Human Populations Exposed to Mutagenic and Re- productive Hazards. White Plains, NY: March of Dimes Foundation. Bonde JP, Giwercman A. 1995. Occupational hazards to male fecundity. Reproductive Medicine Review 4:59â73. Boverhof DR, Burgoon LD, Williams KJ, Zacharewski TR. 2008. Inhibition of estrogen-mediated uterine gene expression responses by dioxin. Molecular Pharmacology 73(1):82â93. Bretveld RW, Thomas CMG, Scheepers PTJ, Zielhuis GA, Roeleveld N. 2006a. Pesticide exposure: The hormonal function of the female reproductive system disrupted? Reproductive Biology and Endocrinology 4:20. Bretveld RW, Thomas CM, Scheepers PT, Zielhuis GA, Roeleveld N. 2006b. Pesticide exposure: The hormonal function of the female reproductive system disrupted? Reproductive Biology and Endocrinology 4:30. 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):1623â1629. Bruner-Tran KL, Rier SE, Eisenberg E, Osteen KG. 1999. The potential role of environmental toxins in the pathophysiology of endometriosis. Gynecologic and Obstetric Investigation 48(1):45â52. Bruner-Tran KL, Yeaman GR, Crispens MA, Igarashi TM, Osteen KG. 2008. Dioxin may promote inflammation-related development of endometriosis. Fertility and Sterility 89(Supplement 5): 1287â1298.
REPRODUCTIVE EFFECTS AND IMPACTS ON FUTURE GENERATIONS 497 Bryce R. 1991. The epidemiology of preterm birth. In: Kiely M, ed. Reproductive and Perinatal Epidemiology. Boca Raton, FL: CRC Press. Pp. 437â444. 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 Childrensâ Cancer Study Group. Cancer Research 49(14):4030â4037. Bulun SE, Zeitoun KM, Kilic G. 2000. Expression of dioxin-related transactivating factors and target genes in human eutopic endometrial and endometriotic tissues. American Journal of Obstetrics & Gynecology 182(4):767â775. Bursian SJ, Beckett KJ, Yamini B, Martin PA, Kannan K, Shields KL, Mohr FC. 2006a. Assessment of effects in mink caused by consumption of carp collected from the Saginaw River, Michigan, USA. Archives of Environmental and Contaminated Toxicology 50(4):614â623. Bursian SJ, Sharma C, Aulerich RJ, Yamini B, Mitchell RR, Beckett KJ, Orazio CE, Moore D, Svirsky S, Tillitt DE. 2006b. Dietary exposure of mink (Mustela vison) to fish from the Housatonic River, Berkshire County, Massachusetts, USA: Effects on organ weights and histology and hepatic concentrations of polychlorinated biphenyls and 2,3,7,8-tetrachlorodibenzo-p-dioxin toxic equivalence. Environmental Toxicology and Chemistry 25(6):1541â1550. Carbone P, Giordano F, Nori F, Mantovani A, Taruscio D, Lauria L, Figa-Talamanca I. 2007. The possible role of endocrine disrupting chemicals in the aetiology of cryptorchidism and hypospa- dias: A population-based caseâcontrol study in rural Sicily. International Journal of Andrology 30(1):3â13. Carmelli D, Hofherr L, Tomsic J, Morgan RW. 1981. A CaseâControl Study of the Relationship Be- tween Exposure to 2,4-D and Spontaneous Abortions in Humans. SRI International. Prepared for the National Forest Products Association and the US Department of Agriculture, Forest Service. Carozza SE, Li B, Elgethun K, Whitworth R. 2008. Risk of childhood cancers associated with resi- dence in agriculturally intense areas in the United States. Environmental Health Perspectives 116(4):559â565. CDC (Centers for Disease Control and Prevention). 1989a. Health Status of Vietnam Veterans Vols I-V, Supplements A-C. Vietnam Experience Study. 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. CDC. 2000. National Center for Health Statistics. National Vital Statistics System. Vital Statistics of the United States, Vol. II, Mortality, Part A, for Data Years 1950â1993. Washington, DC: US Government Printing Office. Data for 1994 to 1998, data are available on the NCHS Web site at http://www.cdc.gov/nchs/datawh/statab/unpubd/mortabs.htm. Chang SF, Sun YY, Yang LY, Hu SY, Tsai SY, Lee WS, Lee YH. 2005. Bcl-2 gene family expression in the brain of rat offspring after gestational and lactational dioxin exposure. Annals of the New York Academy of Sciences. 1042:471â480. Chao HR, Wang YF, Chen HT, Ko YC, Chang EE, Huang YJ, Tsai FY, Tsai CH, Wu CH, Tsou TC. 2007. Differential effect of arecoline on the endogenous dioxin-responsive cytochrome P450 1A1 and on a stably transfected dioxin-responsive element-driven reporter in human hepatoma cells. Journal of Hazardous Materials 149(1):234â237. Charles JM, Henwood SM, Leeming NM. 1999. Developmental toxicity studies in rats and rabbits and two-generation reproduction study in rats on 4-(2,4-dichlorophenoxy) butyric acid. International Journal of Toxicology 18:177â189. 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):858â867.
498 VETERANS AND AGENT ORANGE: UPDATE 2008 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):31â40. Chia SE, Shi LM. 2002. Review of recent epidemiological studies on paternal occupations and birth defects. Occupational and Environmental Medicine 59(3):149â155. Choi JS, Kim IW, Hwang SY, Shin BJ, Kim SK. 2008. Effect of 2,3,7,8-tetrachlorodibenzo-p-dioxin on testicular spermatogenesis-related panels and serum sex hormone levels in rats. BJU Inter- national 101(2):250â255. Choi SS, Miller MA, Harper PA. 2006. In utero exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin induces amphiregulin gene expression in the developing mouse ureter. Toxicological Sciences 94(1):163â174. Clementi M, Causin R, Marzocchi C, Mantovani A, Tenconi R. 2007. A study of the impact of agricultural pesticide use on the prevalence of birth defects in northeast Italy. Reproductive Toxicology 24(1):1â8. Cok I, Donmez MK, Satiroglu MH, Aydinuraz B, Henkelmann B, Shen H, Kotalik J, Schramm KW. 2008. Concentrations of polychlorinated dibenzo-p-dioxins (PCDDs), polychlorinated dibenzofurans (PCDFs), and dioxin-like PCBs in adipose tissue of infertile men. Archives of Environmental Contamination and Toxicology 55(1):143â152. 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):134â137. 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):8â15. Cummings AM, Metcalf JL, Birnbaum L. 1996. Promotion of endometriosis by 2,3,7,8-tetrachloroÂ dibenzo-p-dioxin in rats and mice: Time-dose dependence and species comparison. Toxicology and Applied Pharmacology 138:131â139. Cutfield WS, Hofman PL, Mitchell M, Morison IM. 2007. Could epigenetics play a role in the devel- opmental origins of health and disease? Pediatric Research 61:68Râ75R. Damgaard IN, Main KM, Toppari J, Skakkebaek NE. 2002. Impact of exposure to endocrine dis- rupters in utero and in childhood on adult reproduction. Best Practice and Research Clinical Endocrinology and Metabolism 16(2):289â309. Daniels JL, Olshan AF, Teschke K, Herz-Picciotto I, Savitz DA, Blatt J, Bondy ML, Neglia JP, Pollock BH, Cohn SL, Look AT, Seeger RC, Castleberry RP. 2001. Residential pesticide expo- sure and neuroblastoma. Epidemiology 12:20â27. De Felip E, Porpora MG, di Domenico A, Ingelido AM, Cardelli M, Cosmi EV, Donnez J. 2004. Dioxin-like compounds and endometriosis: A study on Italian and Belgian women of reproduc- tive age. Toxicology Letters 150(2):203â209. del Rio Gomez I, Marshall T, Tsai P, Shao Y-S, Guo YL. 2002. Number of boys born to men exposed to polychlorinated byphenlys [sic]. The Lancet 360:143â144. 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):1457â1475. Dhooge W, van Larebeke N, Koppen G, Nelen V, Schoeters G, Vlietinck R, Kaufman JM, Comhaire F, Flemish E, Health Study G. 2006. Serum dioxin-like activity is associated with reproductive parameters in young men from the general Flemish population. Environmental Health Perspec- tives 114(11):1670â1676. Dienhart MK, Sommer RJ, Peterson RE, Hirshfield AN, Silbergeld EK. 2000. Gestational exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin induces developmental defects in the rat vagina. Toxico- logical Sciences 56:141â149.
REPRODUCTIVE EFFECTS AND IMPACTS ON FUTURE GENERATIONS 499 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):267â273. Donovan JW, MacLennan R, Adena M. 1984. Vietnam service and the risk of cogenital anomalies: A caseâcontrol study. Medical Journal of Australia 140(7):394â397. Driscoll R, Donovan B, Esswein E, Mattorano D. 1998. Health hazard evaluation report. US Depart- ment of Agriculture 1â72. Edwards TM, Myers JP. 2007. Environmental exposures and gene regulation in disease etiology. Environmental Health Perspectives 115(9):1264â1270. Egeland GM, Sweeney MH, Fingerhut MA, Wille KK, Schnorr TM, Halperin WE. 1994. Total serum testosterone and gonadotropins in workers exposed to dioxin. American Journal of Epidemiol- ogy 139:272â281. Ehrlich M. 2003. Expression of various genes is controlled by DNA methylation during mammalian development. Journal of Cellular Biochemistry 88:899â910. Ergaz Z, Avgil M, Ornoy A. 2005. Intrauterine growth restrictionâetiology and consequences: What do we know about the human situation and experimental animal models? Reproductive Toxicol- ogy 20(3):301â322. Erickson J, Mulinare J, Mcclain P, Fitch T, James L, McClearn A, Adams M. 1984a. Vietnam Veter- ansâ 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 veteransâ risks for fathering babies with birth defects. Journal of the American Medical Association 252(7):903â912. Eskenazi B, Mocarelli P, Warner M, Samuels S, Vercellini P, Olive D, Needham LL, Patterson DG Jr, Brambilla P, Gavoni N, Casalini S, Panazza S, Turner W, Gerthoux PM. 2002. Serum dioxin concentrations and endometriosis: A cohort study in Seveso, Italy. Environmental Health Per- spectives 110(7):629â634. Eskenazi B, Mocarelli P, Warner M, Chee WY, Gerthoux PM, Samuels S, Needham LL, Patterson DG Jr. 2003. Maternal serum dioxin levels and birth outcomes in women of Seveso, Italy. En- vironmental Health Perspectives 111(7):947â953. Eskenazi B, Warner M, Marks AR, Samuels S, Gerthoux PM, Vercellini P, Olive DL, Needham L, Patterson D Jr, Mocarelli P. 2005. Serum dioxin concentrations and age at menopause. Environ- mental Health Perspectives 113(7):858â862. Eskenazi B, Warner M, Samuels S, Young J, Gerthoux PM, Needham L, Patterson D, Olive D, Gavoni N, Vercellini P, Mocarelli P. 2007. Serum dioxin concentrations and risk of uterine leiomyoma in the Seveso Womenâs Health Study. American Journal of Epidemiology 166(1):79â87. Farr SL, Cooper GS, Cai J, Savitz DA, Sandler DP. 2004. Pesticide use and menstrual cycle charac- teristics among premenopausal women in the Agricultural Health Study. American Journal of Epidemiology 160(12):1194â1204. Farr SL, Cai J, Savitz DA, Sandler DP, Hoppin JA, Cooper GS. 2006. Pesticide exposure and tim- ing of menopause: The Agricultural Health Study. American Journal of Epidemiology 163(8): 731â742. Felix JF, Van Dooren MF, Klaassens M, Hop WCJ, Torfs CP, Tibboel D. 2008. Environmental factors in the etiology of esophageal atresia and congenital diaphragmatic hernia: Results of a caseâcontrol study. Birth Defects Research Part AâClinical and Molecular Teratology 82(2):98â105. 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 48:157â160.
500 VETERANS AND AGENT ORANGE: UPDATE 2008 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):63â74. Field B, Kerr C. 1988. Reproductive behaviour and consistent patterns of abnormality in offspring of Vietnam veterans. Journal of Medical Genetics 25:819â826. Fierens S, Mairesse H, Heilier JF, de Burbure C, Focant JF, Eppe G, de Pauw E, Bernard A. 2003. Dioxin/polychlorinated biphenyl body burden, diabetes and endometriosis: Findings in a population-based study in Belgium. Biomarkers 8(6):529â534. 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 44:214â221. 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):631â635. Fujita H, Samejima H, Kitagawa N, Mitsuhashi T, Washio T, Yonemoto J, Tomita M, Takahashi T, Kosaki K. 2006. Genome-wide screening of dioxin-responsive genes in fetal brain: Bioinfor- matic and experimental approaches. Congenital Anomalies 46(3):135â143. Fujiwara K, Yamada T, Mishima K, Imura H, Sugahara T. 2008. Morphological and immunohis- tochemical studies on cleft palates induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin in mice. Congenital Anomalies 48(2):68â73. Gao Y, Sahlberg C, Kiukkonen A, Alaluusua S, Pohjanvirta R, Tuomisto J, Lukinmaa PL. 2004. Lacta- tional exposure of Han/Wistar rats to 2,3,7,8-tetrachlorodibenzo-p-dioxin interferes with enamel maturation and retards dentin mineralization. Journal of Dental Research 83(2):139â144. GarcÃa AM, Benavides FG, Fletcher T, Orts E. 1998. Paternal exposure to pesticides and congenital malformations. Scandinavian Journal of Work, Environment and Health 24(6):473â480. Garry VF, Schreinemachers D, Harkins ME, Griffith J. 1996. Pesticide appliers, biocides, and birth defects in rural Minnesota. Environmental Health Perspectives 104(4):394â399. Gray L, Ostby J, Kelce WR. 1997a. A dose response analysis of reproductive effects of a single ges- tational dose of 2,3,7,8-tetrachlorodibenzo-p-dioxin in male Long Evans Hooded rat offspring. Toxicology and Applied Pharmacology 146:11â20. Gray L, Wolf C, Mann P, Ostby J. 1997b. In utero exposure to low doses of 2,3,7,8-tetrachlorodibenzo- p-dioxin alters reproductive development of female Long Evans hooded rat offsprings. Toxicol- ogy and Applied Pharmacology 146:237â244. Greenlee AR, Arbuckle TE, Chyou PH. 2003. Risk factors for female infertility in an agricultural region. Epidemiology 14(4):429â436. Gupta VK, Ali I, Suhas, Saini VK. 2006. Adsorption of 2,4-D and carbofuran pesticides using fertil- izer and steel industry wastes. Journal of Colloid and Interface Science 299(2):556â563. Hanify JA, Metcalf P, Nobbs CL, Worsley KJ. 1981. Aerial spraying of 2,4,5-T and human birth malformations: An epidemiological investigation. Science 212:349â351. 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):56â60. Heacock H, Hertzman C, Demers PA, Gallagher R, Hogg RS, Teschke K, Hershler R, Bajdik CD, Dimich-Ward H, Marion SA, Ostry A, Kelly S. 2000. Childhood cancer in the offspring of male sawmill workers occupationally exposed to chlorophenate fungicides. Environmental Health Perspectives 108(6):499â503. Heiden TCK, Struble CA, Rise ML, Hessner MJ, Hutz RJ, Carvan IMJ. 2008. Molecular targets of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) within the zebrafish ovary: Insights into TCDD- induced endocrine disruption and reproductive toxicity. Reproductive Toxicology 25(1):47â57. Heilier JF, Nackers F, Verougstraete V, Tonglet R, Lison D, Donnez J. 2005. Increased dioxin-like compounds in the serum of women with peritoneal endometriosis and deep endometriotic (ad- enomyotic) nodules. Fertility and Sterility 84(2):305â312.
REPRODUCTIVE EFFECTS AND IMPACTS ON FUTURE GENERATIONS 501 Heilier JF, Donnez J, Defrere S, Van Kerckhove V, Donnez O, Lison D. 2006. Serum dioxin-like compounds and aromatase (CYP19) expression in endometriotic tissues. Toxicology Letters 167(3):238â244. Heilier JF, Donnez J, Nackers F, Rousseau R, Verougstraete V, Rosenkranz K, Donnez O, Grandjean F, Lison D, Tonglet R. 2007. Environmental and host-associated risk factors in endometrio- sis and deep endometriotic nodules: A matched caseâcontrol study. Environmental Research 103(1):121â129. Henriksen GL, Michalek JE, Swaby JA, Rahe AJ. 1996. Serum dioxin, testosterone, and gonadotro- pins in veterans of Operation Ranch Hand. Epidemiology 7(4):352â357. Hertz-Picciotto I, Samuels SJ. 1988. Incidence of early loss of pregnancy. New England Journal of Medicine 319(22):483â484. Hertz-Picciotto I, Jusko TA, Willman EJ, Baker RJ, Keller JA, Teplin SW, Charles MJ. 2008. A cohort study of in utero polychlorinated biphenyl (PCB) exposures in relation to secondary sex ratio. Environmental Health: A Global Access Science Source 7:37. Ho HM, Ohshima K, Watanabe G, Taya K, Strawn EY, Hutz RJ. 2006. TCDD increases inhibin a production by human luteinized granulosa cells in vitro. Journal of Reproduction and Develop- ment 52(4):523â528. Hojo R, Zareba G, Kai JW, Baggs RB, Weiss B. 2006. Sex-specific alterations of cerebral cortical cell size in rats exposed prenatally to dioxin. Journal of Applied Toxicology 26(1):25â34. Hutt KJ, Shi Z, Albertini DF, Petroff BK. 2008. The environmental toxicant 2,3,7,8-tetrachlorodibenzo- p-dioxin disrupts morphogenesis of the rat pre-implantation embryo. BMC Developmental Biology 8(1). Igarashi TM, Bruner-Tran KL, Yeaman GR, Lessey BA, Edwards DP, Eisenberg E, Osteen KG. 2005. Reduced expression of progesterone receptor-B in the endometrium of women with endome- triosis and in cocultures of endometrial cells exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin. Fertility and Sterility 84(1):67â74. Ikeda M, Tamura M, Yamashita J, Suzuki C, Tomita T. 2005b. Repeated in utero and lactational 2,3,7,8-tetrachlorodibenzo-p-dioxin exposure affects male gonads in offspring, leading to sex ratio changes in F2 progeny. Toxicology and Applied Pharmacology 206(3):351â355. Ilvesaro J, Pohjanvirta R, Tuomisto J, Viluksela M, Tuukkanen J. 2005. Bone resorption by aryl hydrocarbon receptor-expressing osteoclasts is not disturbed by TCDD in short-term cultures. Life Sciences 77(12):1351â1366. Infante-Rivard C, Labuda D, Krajinovic M, Sinnett D. 1999. Risk of childhood leukemia associated with exposure to pesticides and with gene polymorphisms. Epidemiology 10:481â487. 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. 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. Jaenisch R, Bird A. 2003. Epigenetic regulation of gene expression: How the genome integrates intrinsic and environmental signals. Nature Genetics 33:245â354. James WH. 2006. Offspring sex ratios at birth as markers of paternal endocrine disruption. Environ- mental Research 100:77â85.
502 VETERANS AND AGENT ORANGE: UPDATE 2008 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):303â309. 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):280â283. Jiang YH, Bressler J, Beaudet AL. 2004. Epigenetics and human disease. Annual Review of Genomics and Human Genetics 5:479â510. John-Greene JA, Ouellette JH, Jeffries TK, Johnson KA, Rao KS. 1985. Teratological evaluation of picloram potassium salt in rabbits. Food and Chemical Toxicology 23(8):753â756. Johnson KL, Cummings AM, Birnbaum LS. 1997. Promotion of endometriosis in mice by polychlo- rinated dibenzo-p-dioxins, dibenzofurans, and biphenyls. Environmental Health Perspectives 105(7):750â755. Kallen B. 1988. Epidemiology of Human Reproduction. Boca Raton, FL: CRC Press. Kalter H, Warkany J. 1983. Congenital malformations. Etiologic factors and their role in prevention (first of two parts). New England Journal of Medicine 308:424â431. Kang HK, Mahan CM, Lee KY, Magee CA, Mather SH, Matanoski G. 2000. Pregnancy out- comes among US women Vietnam veterans. American Journal of Industrial Medicine 38(4): 447â454. Karmaus W, Huang S, Cameron L. 2002 Parental concentration of dichlorodiphenyl dichloroethene and polychlorinated biphenyls in Michigan fish eaters and sex ratio in offspring. Journal of Occupational and Environmental Medicine 44(1):8â13. Keller JM, Allen DE, Davis CR, Leamy LJ. 2007a. 2,3,7,8-Tetrachlorodibenzo-p-dioxin affects fluc- tuating asymmetry of molar shape in mice, and an epistatic interaction of two genes for molar size. Heredity 98(5):259â267. Keller JM, Huang JC, Huet-Hudson Y, Leamy LJ. 2007b. The effects of 2,3,7,8-tetrachlorodibenzo- p-dioxin on molar and mandible traits in congenic mice: A test of the role of the Ahr locus. Toxicology 242(1-3):52â62. Keller JM, Huet-Hudson YM, Leamy LJ. 2007c. Qualitative effects of dioxin on molars vary among inbred mouse strains. Archives of Oral Biology 52(5):450â454. Keller JM, Huet-Hudson Y, Leamy LJ. 2008. Effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin on molar development among non-resistant inbred strains of mice: A geometric morphometric analysis. Growth, Development, and Aging 71(1):3â16. Kerr M, Nasca PC, Mundt KA, Michalek AM, Baptiste MS, Mahoney MC. 2000. Parental occu- pational exposures and risk of neuroblastoma: A caseâcontrol study (United States). Cancer Causes and Control 11:635â643. Khorram O, Garthwaite M, Golos T. 2002. Uterine and ovarian aryl hydrocarbon receptor (ahr) and aryl hydrocarbon receptor nuclear translocator (arnt) mrna expression in benign and malignant gynaecological conditions. Molecular Human Reproduction 8(1):75â80. Khorram O, Garthwaite M, Jones J, Golos T. 2004. Expression of aryl hydrocarbon receptor (AHR) and aryl hydrocarbon receptor nuclear translocator (ARNT) mRNA expression in human sper- matozoa. Medical Science Monitor 10(5):BR135âBR138. Kline J, Stein Z, Susser M. 1989. Conception to Birth: Epidemiology of Prenatal Development. New York: Oxford University Press. Knobil E, Neill JD, Greenwald GS, Markert CL, Pfaff DW, eds. 1994. The Physiology of Reproduc- tion. New York: Raven Press. Koopman-Esseboom C, Huisman M, Weisglas-Kuperus N, Van der Paauw CG, Tuinstra L, Boersma ER, Sauer PJJ. 1994. PCB, dioxin levels in plasma and human milk of 418 Dutch women and their infants. Predictive value of PCB congener levels in maternal plasma for fetal and infantâs exposure to PCBs and dioxins. Chemosphere 28:1721â1732.
REPRODUCTIVE EFFECTS AND IMPACTS ON FUTURE GENERATIONS 503 Kransler KM, McGarrigle BP, Russell RJ, Olson JR. 2008. Effects of Helicobacter infection on developmental toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin in Holtzman rats. Lab Animal 37(4):171â175. 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):39â50. Kristensen P, Irgens LM, Andersen A, Bye AS, Sundheim L. 1997. Birth defects among offspring of Norwegian farmers, 1967â1991. Epidemiology 8(5):537â544. Kuroda M, Oikawa K, Ohbayashi T, Yoshida K, Yamada K, Mimura J, Matsuda Y, Fujii-Kuriyama Y, Mukai K. 2005. A dioxin sensitive gene, mammalian WAPL, is implicated in spermatogenesis. FEBS Letters 579(1):167â172. Lacasana M, Vazquez-Grameix H, Borja-Aburto VH, Blanco-Munoz J, Romieu I, Aguilar-Garduno C, Garcia AM. 2006. Maternal and paternal occupational exposure to agricultural work and the risk of anencephaly. Occupational and Environmental Medicine 63(10):649â656. Lahvis GP, Lindell SL, Thomas RS, McCuskey RS, Murphy C, Glover E, Bentz M, Southard J, Bradfield CA. 2000. Portosystemic shunting and persistent fetal vascular structures in aryl hydrocarbon receptorâdeficient mice. Proceedings of the National Academy of Sciences of the United States of America 97(19):10442â10447. 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):835â844. Landrigan P, Garg A, Droller DBJ. 2003. Assessing the effects of endocrine disruptors in the National Childrenâs Study. Environmental Health Perspectives 111(13):1678â1682. Larsen SB, Joffe M, Bonde JP. 1998. Time to pregnancy and exposure to pesticides in Danish farmers. Occupational and Environmental Medicine 55(4):278â283. 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):1403â1408. Lerda D, Rizzi R. 1991. Study of reproductive function in persons occupationally exposed to 2,4- dichlorophenoxyacetic acid (2,4-D). Mutation Research 262(1):47â50. Lind PM, Eriksen EF, Sahlin L, Edlund M, Ãrberg J. 1999. Effects of the antiestrogenic environmental pollutant 3,3â²,4,4â²,5-pentachlorobiphenyl (PCB 126) in rat bone and uterus: Diverging effects in ovariectomized and intact animals. Toxicology and Applied Pharmacology 154(3):236â244. Lind PM, Larsson S, Oxlund H, Hakansson H, Nyberg K, Eklund T, Ãrberg J. 2000a. Change in bone tissue composition and impaired bone strength in rats exposed to 3,3â²,4,4â²,5-pentachlorobiphenyl (PCB 126). Toxicology 150:41â51. Lind PM, Ãrberg J, Edlund U-B, SjÃ¶blom L, Lind L. 2000b. Bone tissue composition, dimensions and strength in rats an increased dietary level of vitamin A or exposed to 3,3â²,4,4â²,5-Âpentachlorobiphenyl (PCB 126) alone or in combination with vitamin C. Toxicology 151:11â23. 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):529â536. Lorber M, Phillips L. 2002. Infant exposure to dioxin-like compounds in breast milk. Environmental Health Perspectives 110(6):A325âA332. Lucifero D, Mann MR, Bartolomei MS, Trasler JM. 2004. Gene-specific timing and epigenetic memory in oocyte imprinting. Human Molecular Genetics 13:839â849. Mably TA, Moore RW, Goy RW, Peterson RE. 1992. In utero and lactational exposure of male rats to 2,3,7,8-tetrachlorodibenzo-p-dioxin. 2. Effects on sexual behavior and the regulation of lutein- izing hormone secretion in adulthood. Toxicology and Applied Pharmacology 114:108â117.
504 VETERANS AND AGENT ORANGE: UPDATE 2008 Mastroiacovo P, Spagnolo A, Marni E, Meazza L, Betrollini R, Segni G, Brogna-Pignatti C. 1988. Birth deffects in Seveso area after TCDD contamination. Journal of American Medical Associa- tion 259:1668â1672 (published erratum appears in JAMA 1988, 260:792). Mayani A, Barel S, Soback S, Almagor M. 1997. Dioxin concentrations in women with endometriosis. Human Reproduction 12(2):373â375. Mehta V, Peterson RE, Heideman W. 2008. 2,3,7,8-Tetrachlorodibenzo-p-dioxin exposure prevents cardiac valve formation in developing zebrafish. Toxicological Sciences 104(2):303â311. Meinert R, SchÃ¼z J, Kaletsch U, Kaatsch P, Michaelis J. 2000. Leukemia and non-Hodgkinâs lym- phoma in childhood and exposure to pesticides: Results of a register-based caseâcontrol study in Germany. American Journal of Epidemiology 151(7):639â646. 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): 1589â1595. 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 HerbicidesâReproductive Outcome Update. US Department of Commerce, National Technical Information Service. Report number AFRL-HE-BR-TR-1998-0073. Michalek JE, Rahe AJ, Boyle CA. 1998b. Paternal dioxin, preterm birth, intrauterine growth retarda- tion, and infant death. Epidemiology 9(2):161â167. Miettinen HM, Pulkkinen P, Jamsa T, Koistinen J, Simanainen U, Tuomisto J, Tuukkanen J, Viluksela M. 2005. Effects of in utero and lactational TCDD exposure on bone development in differen- tially sensitive rat lines. Toxicological Sciences 85(2):1003â1012. Miettinen HM, Sorvari R, Alaluusua S, Murtomaa M, Tuukkanen J, Viluksela M. 2006. The effect of perinatal TCDD exposure on caries susceptibility in rats. Toxicological Sciences 91(2): 568â575. Mitsui T, Sugiyama N, Maeda S, Tohyama C, Arita J. 2006. Perinatal exposure to 2,3,7,8-tetrachlorod- ibenzo-p-dioxin suppresses contextual fear conditioning-accompanied activation of cyclic AMP response element-binding protein in the hippocampal CA1 region of male rats. Neuroscience Letters 398(3):206â210. Mocarelli P, Brambilla P, Gerthoux PM, Patterson DG Jr, Needham LL. 1996. Change in sex ratio with exposure to dioxin. Lancet 348(9024):409. Mocarelli P, Gerthoux PM, Ferrari E, Patterson DG Jr, Kieszak SM, Brambilla P, Vincoli N, Signorini S, Tramacere P, Carreri V, Sampson EJ, Turner WE, Needham LL. 2000. Paternal concentrations of dioxin and sex ratio of offspring. The Lancet 355:1858â1863. Mocarelli P, Gerthoux PM, Patterson DG Jr, Milani S, Limonta G, Bertona M, Signorini S, Tramacere P, Colombo L, Crespi C, Brambilla P, Sarto C, Carreri V, Sampson EJ, Turner WE, Needham LL. 2008. Dioxin exposure, from infancy through puberty, produces endocrine disruption and affects human semen quality. Environmental Health Perspectives 116(1):70â77. 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):293â303. Morgan HD, Santos F, Green K, Dean W, Reik W. 2005. Epigenetic reprogramming in mammals. Human Molecular Genetics 14:R47âR58. 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):161â182. Moshammer H, Neuberger M. 2000. Sex ratio in the children of the Austrian chloracne cohort. The Lancet 356:1271.
REPRODUCTIVE EFFECTS AND IMPACTS ON FUTURE GENERATIONS 505 Nagasaka T, Koi M, Kloor M, Gebert J, Vilkin A, Nishida N, Shin SK, Sasamoto H, Tanaka N, Matsubara N, Boland CR, Goel A. 2008. Mutations in both KRAS and BRAF may contribute to the methylator phenotype in colon cancer. Gastroenterology 134:1950â1960. Nakajima S, Saijo Y, Kato S, Sasaki S, Uno A, Kanagami N, Hirakawa H, Hori T, Tobiishi K, Todaka T, Nakamura Y, Yanagiya S, Sengoku Y, Iida T, Sata F, Kishi R. 2006. Effects of prenatal ex- posure to polychlorinated biphenyls and dioxins on mental and motor development in Japanese children at 6 months of age. Environmental Health Perspectives 114(5):773â778. Nayyar T, Wu J, Hood DB. 2003. Downregulation of hippocampal NMDA receptor expression by prenatal exposure to dioxin. Cellular and Molecular Biology 49(8):1357â1362. NCI (National Cancer Institute). 2001. Surveillance, Epidemiology, and End Results (SEER) data- base. http://seer.cancer.gov/ScientificSystems/CanQues (Accessed March 19). Negishi T, Shimomura H, Koyama T, Kawasaki K, Ishii Y, Kyuwa S, Yasuda M, Kuroda Y, Yoshikawa Y. 2006. Gestational and lactational exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin affects social behaviors between developing rhesus monkeys (Macaca mulatta). Toxicology Letters 160(3):233â244. NICHD (National Institute of Child Health and Human Development). 2007. Endometriosis. Na- tional Institute of Health. http://www.nichd.nih.gov/health/topics/endometriosis.cfm (Accessed December 17, 2008). Nishijo M, Tawara K, Nakagawa H, Honda R, Kido T, Nishijo H, Saito S. 2008. 2,3,7,8-TetrachloroÂ dibenzo-p-dioxin in maternal breast milk and newborn head circumference. Journal of Exposure Science and Environmental Epidemiology 18(3):246â251. Nishimura N, Matsumura F, Vogel CFA, Nishimura H, Yonemoto J, Yoshioka W, Tohyama C. 2008. Critical role of cyclooxygenase-2 activation in pathogenesis of hydronephrosis caused by lacta- tional exposure of mice to dioxin. Toxicology and Applied Pharmacology 231(3):374â383. Oh E, Lee E, Im H, Kang HS, Jung WW, Won NH, Kim EM, Sul D. 2005. Evaluation of immuno- and reproductive toxicities and association between immunotoxicological and genotoxicological parameters in waste incineration workers. Toxicology 210(1):65â80. Ohsako S, Miyabara Y, Sakaue M, Ishimura R, Kakeyama M, Izumi H, Yonemoto J, Tohyama C. 2002. Developmental stageâspecific effects of perinatal 2,3,7,8-tetrachlorodibenzo-p-dioxin exposure on reproductive organs of male rat offspring. Toxicological Sciences 66(2):283â292. Ohtake F, Baba A, Fujii-Kuriyama Y, Kato S. 2008. Intrinsic AhR function underlies cross-talk of dioxins with sex hormone signalings. Biochemical and Biophysical Research Communications 370(4):541â546. Oikawa K, Ohbayashi T, Mimura J, Fujii-Kuriyama Y, Teshima S, Rokutan K, Mukai K, Kuroda M. 2002. Dioxin stimulates synthesis and secretion of IgE-dependent histamine-releasing factor. Biochemical and Biophysical Research Communications 290(3):984â987. Oikawa K, Kosugi Y, Ohbayashi T, Kameta A, Isaka K, Takayama M, Kuroda M, Mukai K. 2003. Increased expression of IgE-dependent histamine-releasing factor in endometriotic implants. Journal of Pathology 199(3):318â323. 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):361â364. Park JS, Hwang SY, Hwang BY, Han K. 2008. The spermatogenic effect of 50% ethanol extracts of Yacon and its ameliorative effect against 2,3,7,8-tetrachlorodibenzo-p-dioxin induced testicular toxicity in the rat. Natural Product Sciences 14(2):73â80. Pauwels A, Schepens PJC, Hooghe TD, Delbeke L, Dhont M, Brouwer A, Weyler J. 2001. The risk of endometriosis and exposure to dioxins and polychlorinated biphenyls: A caseâcontrol study of infertile women. Human Reproduction 16(10):2050â2055. Pearce MS, Parker L. 2000. Paternal employment in agriculture and childhood kidney cancer. Pedi- atric Hematology and Oncology 17(3):223â230.
506 VETERANS AND AGENT ORANGE: UPDATE 2008 Pearce MS, Hammal DM, Dorak MT, McNally RJ, Parker L. 2006. Paternal occupational expo- sure to pesticides or herbicides as risk factors for cancer in children and young adults: A caseâcontrol study from the North of England. Archives of Environmental and Occupational Health 61(3):138â144. Peltier MR. 2003. Immunology of term and preterm labor. Reproductive Biology and Endocrinology 1:122â132. Pesatori AC, Consonni D, Tironi A, Zocchetti C, Fini A, Bertazzi PA. 1993. Cancer in a young popula- tion in a dioxin-contaminated area. International Journal of Epidemiology 22(6):1010â1013. Petrelli G, Figa-Talamanca I, Tropeano R, Tangucci M, Cini C, Aquilini S, Gasperini L, Meli P. 2000. Reproductive male-mediated risk: Spontaneous abortion among wives of pesticide applicators. European Journal of Epidemiology 16(4):391â393. Polsky JY, Aronson KJ, Heaton JP, Adams MA. 2007. Pesticides and polychlorinated biphenyls as potential risk factors for erectile dysfunction. Journal of Andrology 28(1):28â37. Porpora MG, Ingelido AM, di Domenico A, Ferro A, Crobu M, Pallante D, Cardelli M, Cosmi EV, De Felip E. 2006. Increased levels of polychlorobiphenyls in Italian women with endometriosis. Chemosphere 63(8):1361â1367. Ray SS, Swanson HI. 2004. Dioxin-induced immortalization of normal human keratinocytes and silencing of p53 and p16INK4a. Journal of Biological Chemistry 279(26):27187â27193. Reik W, Walter J. 2001. Genomic imprinting: Parental influence on the genome. Nature Review Genetics 2:21â32. Reik W, Dean W, Walter J. 2001. Epigenetic reprogramming in mammalian development. Science 293:1089â1093. 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):951â966. 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):93â100. Rier SE, Martin DC, Bowman RE, Dmowski WP, Becker JL. 1993. Endometriosis in rhesus monkeys (Macaca mulatta) following chronic exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin. Funda- mental and Applied Toxicology 21(4):433â441. Romo A, Carceller R, Tobajas J. 2009. Intrauterine growth retardation (IUGR): Epidemiology and etiology. Pediatric Endocrinology Reviews 6(Supplement 3):332â336. Roundtree MR, Bachman KE, Herman JG, Baylin SB. 2001. DNA methylation, chromatin inheri- tance, and cancer. Oncogene 20:3156â3165. 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):1787â1793. Ryan JJ, Amirova Z, Carrier G. 2002. Sex ratios of children of Russian pesticide producers exposed to dioxin. Environmental Health Perspectives 110(11):A699âA701. Sagiv SK, Tolbert PE, Altshul LM, Korrick SA. 2007. Organochlorine exposures during pregnancy and infant size at birth. Epidemiology 18(1):120â129. Savitz DA, Arbuckle A, Kaczor D, Curtis KM. 1997. Male pesticide exposure and pregnancy out- come. American Journal of Epidemiology 146(12):1025â1036. Schnorr TM, Lawson CC, Whelan EA, Dankovic DA, Deddens JA, Piacitelli LA, Reefhuis J, Sweeney MH, Connally LB, Fingerhut MA. 2001. Spontaneous abortion, sex ratio, and paternal occupational exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin. Environmental Health Perspec- tives 109(11):1127â1132. Schreinemachers DM. 2003. Birth malformations and other adverse perinatal outcomes in four US wheat-producing states. Environmental Health Perspectives 111(9):1259â1264.
REPRODUCTIVE EFFECTS AND IMPACTS ON FUTURE GENERATIONS 507 Schultz R, Suominen J, Varre T, Hakovirta H, Parvinen M, Toppari J, Pelto-Huikko M. 2003. Expres- sion of aryl hydrocarbon receptor and aryl hydrocarbon receptor nuclear translocator messenger ribonucleic acids and proteins in rat and human testis. Endocrinology 144(3):767â776. Schwartz LS. 1998. Health Problems of Women Veterans of the Vietnam War. Doctoral dissertation, Yale University. Shi Z, Valdez KE, Ting AY, Franczak A, Gum SL, Petroff BK. 2007. Ovarian endocrine disruption underlies premature reproductive senescence following environmentally relevant chronic ex- posure to the aryl hydrocarbon receptor agonist 2,3,7,8-tetrachlorodibenzo-p-dioxin. Biology of Reproduction 76(2):198â202. 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 37:197â200. Staessen JA, Nawrot T, Hond ED, Thijs L, Fagard R, Hoppenbrouwers K, Koppen G, Nelen V, Schoeters G, Vanderschueren D, Van Hecke E, Verschaeve L, Vlietinck R, Roels HA. 2001. Renal function, cytogenetic measurements, and sexual development in adolescents in relation to environmental pollutants: A feasibility study of biomarkers. Lancet 357(9269):1660â1669. [Comment in Lancet 2001. 358(9295):1816â1817.] Stellman SD, Stellman JM, Sommer JF Jr. 1988. Health and reproductive outcomes among American Legionnaires in relation to combat and herbicide exposure in Vietnam. Environmental Research 47:150â174. 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 128:410â419. Suskind RR, Hertzberg VS. 1984. Human health effects of 2,4,5-T and its toxic contaminants. Journal of the American Medical Association 251:2372â2380. 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):1836â1847. Swan SH, Kruse RL, Liu F, Barr DB, Drobnis EZ, Redmon JB, Wang C, Brazil C, Overstreet JW; Study for Future Families Research Group. 2003. Semen quality in relation to biomarkers of pesticide exposure. Environmental Health Perspectives 111(12):1478â1484. Tango T, Fujita T, Tanihata T, Minowa M, Doi Y, Kato N, Kunikane S, Uchiyama I, Tanaka M, Uehata 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):83â93. Tas S, Lauwerys R, Lison D. 1996. Occupational hazards for the male reproductive system. Critical Reviews in Toxicology 26(3):261â307. Teilmann G, Juul A, Skakkebaek NE, Toppari J. 2002. Putative effects of endocrine disrupters on pubertal development in the human. Best Practice and Research Clinical Endocrinology and Metabolism 16(1):105â121. 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):1263â1270. Toft G, Long M, Kruger T, Hjelmborg PS, Bonde JP, Rignell-Hydbom A, Tyrkiel E, Hagmar L, Giwercman A, Spano M, Bizzaro D, Pedersen HS, Lesovoy V, Ludwicki JK, Bonefeld-Jorgensen EC. 2007. Semen quality in relation to xenohormone and dioxin-like serum activity among Inuits and three European populations. Environmental Health Perspectives 115 (Supplement 1):15â20. 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 115:695â713.
508 VETERANS AND AGENT ORANGE: UPDATE 2008 Tsuchiya M, Tsukino H, Iwasaki M, Sasaki H, Tanaka T, Katoh T, Patterson DG Jr, Turner W, Needham L, Tsugane S. 2007. Interaction between cytochrome P450 gene polymorphisms and serum organochlorine TEQ levels in the risk of endometriosis. Molecular Human Reproduction 13(6):399â404. Tsukimori K, Tokunaga S, Shibata S, Uchi H, Nakayama D, Ishimaru T, Nakano H, Wake N, Yoshimura T, Furue M. 2008. Long-term effects of polychlorinated biphenyls and dioxins on pregnancy outcomes in women affected by the Yusho incident. Environmental Health Perspec- tives 116(5):626â630. Tuyet LTN, Johansson A. 2001. Impact of chemical warfare with Agent Orange on womenâs reproduc- tive lives in Vietnam: A pilot study. Reproductive Health Matters 9(18):156â164. Van den Berg M, Birnbaum L, Bosveld AT, Brunstrom B, Cook P, Feeley M, Giesy JP, Hanberg A, Hasegawa R, Kennedy SW, et al. 1998. Toxic equivalency factors (TEFs) for PCBs, PCDDs, PCDFs for humans and wildlife. Environmental Health Perspectives 106:775â792. Vreugdenhil H, Weisglas-Kuperus N. 2000. Effects of environmental exposure to polychlorinated bi- phenyls and dioxins on cognitive development in young children. NeuroToxicology 21(4):620. Vreugdenhil HJI, Slijper FME, Mulder PGH, Weisglas-Duperus N. 2002. Effects of perinatal ex- posure to PCBs and dioxins on play behavior in Dutch children at school age. Environmental Health Perspectives 110(10):A593âA598. Vreugdenhil HJ, Mulder PG, Emmen HH, Weisglas-Kuperus N. 2004. Effects of perinatal exposure to PCBs on neuropsychological functions in the Rotterdam cohort at 9 years of age. NeuroÂ psychology 18(1):185â193. Wang S-L, Chang Y-C, Chao H-R, Li C-M, Li L-A, Lin L-Y, Papke O. 2006. Body burdens of poly- chlorinated dibenzo-p-dioxins, dibenzofurans, and biphenyls and their relations to estrogen metabolism in pregnant women. Environmental Health Perspectives 114(5):740â745. Warner M, Samuels S, Mocarelli P, Gerthoux PM, Needham L, Patterson DG Jr, Eskenazi B. 2004. Serum dioxin concentrations and age at menarche. Environmental Health Perspectives 112(13):1289â1292. Warner M, Eskenazi B, Olive DL, Samuels S, Quick-Miles S, Vercellini P, Gerthoux PM, Needham L, Patterson DG Jr, Mocarelli P. 2007. Serum dioxin concentrations and quality of ovarian function in women of Seveso. Environmental Health Perspectives 115(3):336â340. 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 Epidemiology 151(3):231â240. 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):472â480. Wikiera B, Basiak A, Barg E, Noczynska A. 2007. Precocious thelarcheâCurrent opinions. Advances in Clinical and Experimental Medicine 16(2):329â334. Wilcox AJ, Weinberg CR, OâConnor JF, Baird DD, Schlatterer JP, Canfield RE, Armstrong EG, Nisula BC. 1988. Incidence of early pregnancy loss. New England Journal of Medicine 319: 189â194. 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 6:17â22. 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):1790â1797. Yamada T, Fujiwara K, Mishima K, Imura H, Sugahara T. 2007. Effects of 2,3,7,8-tetrachlorodibenzo- p-dioxin on the development of murine palate in organ culture. Asian Journal of Oral and Maxillofacial Surgery 19(4):185â189.
REPRODUCTIVE EFFECTS AND IMPACTS ON FUTURE GENERATIONS 509 Yamano Y, Ohyama K, Ohta M, Sano T, Ritani A, Shimada J, Ashida N, Yoshida E, Ikehara K, Morishima I. 2005. A novel spermatogenesis related factor-2 (SRF-2) gene expression affected by TCDD treatment. Endocrine Journal 52(1):75â81. Yamauchi M, Kim EY, Iwata H, Shima Y, Tanabe S. 2006. Toxic effects of 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD) in developing red seabream (Pagrus major) embryo: An association of mor- phological deformities with AHR1, AHR2 and CYP1A expressions. Aquatic Toxicology 80(2): 166â179. Yamazaki Y, Mann MR, Lee SS, Marh J, McCarrey JR, Yanagimachi R, Bartolomei MS. 2003. Re- programming of primordial germ cells begins before migration into the genital ridge, making these cells inadequate donors for reproductive cloning. Proceedings of the National Academy of Sciences of the United States of America 100:12207â12212. Yang JZ, Agarwal SK, Foster WG. 2000. Subchronic exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin modulates the pathophysiology of endometriosis in the cynomolgus monkey. Toxicological Sciences 56:374â381. Yasuda I, Yasuda M, Sumida H, Tsusaki H, Arima A, Ihara T, Kubota S, Asaoka K, Tsuga K, Akagawa Y. 2005. In utero and lactational exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) af- fects tooth development in rhesus monkeys. Reproductive Toxicology 20(1):21â30. Ye L, Leung LK. 2008. Effect of dioxin exposure on aromatase expression in ovariectomized rats. Toxicology and Applied Pharmacology 229(1):102â108. Yen SC, Jaffe RB. 1991. Reproductive Endocrinology. Philadelphia: W.B. Saunders Company. Yoshimura T, Kaneko S, Hayabuchi H. 2001. Sex ratio in offspring of those affected by dioxin and dioxin-like compounds: The Yusho, Seveso, and Yucheng incidents. Occupational and Envi- ronmental Medicine 58(8):540â541. Yu J, Wang Y, Zhou W-H, Wang L, He Y-Y, Li D-J. 2008. Combination of estrogen and dioxin is involved in the pathogenesis of endometriosis by promoting chemokine secretion and invasion of endometrial stromal cells. Human Reproduction 23(7):1614â1626. Zhao D, Lebovic DI, Taylor RN. 2002. Long-term progestin treatment inhibits RANTES (regulated on activation, normal T cell expressed and secreted) gene expression in human endometrial stromal cells. Journal of Clinical Endocrinology and Metabolism 87(6):2514â2519.