The Interplay Between Environmental Chemical Exposures and Obesity: Proceedings of a Workshop (2016)

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3 Developmental View of the Role of Chemical Exposures and Obesity In the workshop’s second session, three speakers discussed what is known about the relationship between obesity and exposure to various chemicals. In particular, the session was devoted to epidemiological studies that have examined that relationship. Gwen Collman, the director of the extramural program at the National Institute of Environmental Health Sciences (NIEHS), chaired the session. EFFECT OF PRENATAL EXPOSURE TO ORGANOCHLORINES ON CHILDHOOD OBESITY The first speaker was Dania Valvi, a postdoctoral research fellow at the Harvard T.H. Chan School of Public Health and the Center for Research in Environmental Epidemiology in Barcelona, Spain. In her research, Valvi focuses on whether early life exposures to environmental chemicals influence children, with a special interest in studying obesity and metabolic diseases. Valvi’s presentation detailed evidence from birth cohort studies evaluating the impact of prenatal and postnatal exposures to persistent organic pollutants (POPs) on the development of childhood obesity. In particular, she presented evidence from two studies: the INMA birth cohort 21 

22 ENVIRONMENTAL CHEMICAL EXPOSURES AND OBESITY study from Spain1 and the Children’s Health and the Environment in the Faroes study.2 Persistent Organic Pollutants One group of potentially obesogenic chemical substances that Valvi’s research focuses on is POPs. This group of chemicals includes pesticides, such as dichlorodiphenyltrichloroethane (DDT) and its prime metabolite, dichlorodiphenyldichloroethylene (DDE); hexachlorobenzene; industrial chemicals, including polychlorinated biphenyls (PCBs); and their by-products. The use of POPs was gradually restricted until they were finally banned in developed countries beginning in the 1970s. However, widespread exposure to POPs is still of interest because of their characteristics. In particular, they have a slow biodegradation rate of decades, so they are highly persistent in the environment; they can be transported over long distances in the environment through air and water; and they are highly lipophilic, that is, they have an affinity to and tend to dissolve in fats and therefore can accumulate in animal and human fat tissues and remain there for years. Biomonitoring studies are still reporting detectable concentrations of most of these compounds in the blood of a high percentage of the population—more than 90 percent of subjects examined, including pregnant women and children. The main route by which people are exposed to POPs nowadays is through the food that they eat, particularly foods with high fat contents, such as fatty fish, meat, and dairy products. Furthermore, children are exposed to these chemicals very early in life: before birth through the maternal bloodstream via the placenta and after birth through breast milk. 1 INMA (Infancia y Medio Ambiente) is a Spanish research network focused on studying environmental pollutants in the air, water, and diets of children and how these pollutants affect children’s health, starting during pregnancy and continuing through childhood development until the end of adolescence. For more information, see http://www.proyectoinma.org/presentacion-inma/en_index.html (accessed August 3, 2015). 2 The Children’s Health and the Environment in the Faroes study focuses on the health of children and adults in the Faroe Islands, focusing specifically on the impact of marine contaminants on a population with a seafood-heavy diet. For more information, see http://www.chef-project.dk (accessed March 23, 2016). 

THE ROLE OF CHEMICAL EXPOSURES AND OBESITY 23 POPs are one of the chemical groups that have been hypothesized to cause obesity in humans. Their obesogenic effects have been considered in a growing number of human studies; however, few animal studies aiming to elucidate the effects of these chemicals on obesity are currently being conducted; there is much less evidence from experimental studies for these chemicals than for other emerging chemicals, such as the plasticizers bisphenol A (BPA) and phthalates. Although the mechanisms by which POPs may cause obesity remain unknown, animal studies support their role in weight gain. As an example, Valvi described a recent study on DDT carried out in mice by La Merrill and colleagues (2014). This study found that developmental exposures to DDT increased fat mass and that the effects were mediated through decreases of energy expenditures in females but not in males. The reductions in energy expenditure also became worse after the animals were placed on a high-fat diet. Summarizing evidence from animal studies, Valvi said that even though more work is needed to elucidate the underlying mechanisms, the available evidence seems to suggest that susceptibility may depend on sex and other factors, such as diet. Animal studies have further shown that POPs, like other endocrine disrupters, may exhibit different effects at lower and higher doses of exposure. From the human studies available, most studies are birth cohort studies that evaluated exposures during pregnancy and looked for associations with childhood obesity; almost none of these studies evaluated postnatal exposure. The best studied of the POPs so far are DDE and PCBs. Almost all of the studies have assessed obesity using body mass index (BMI), which is just an indirect measure of adiposity; however, it is of interest to see whether chemical exposures increase fat mass and not just BMI. Overall findings for the association between low- dose DDE exposure and increased BMI are fairly consistent, while the associations for other POPs are less consistent across studies. INMA Birth Cohort Studies Valvi then turned to a description of the INMA birth cohort studies in Spain.3 That country, she noted, has rates of overweight and obesity in children that are among the highest in Europe. 3 For more information, see http://www.proyectoinma.org/en_index.html (accessed March 23, 2016). 

24 ENVIRONMENTAL CHEMICAL EXPOSURES AND OBESITY INMA is a network of seven birth cohort studies: the oldest three cohorts in the geographical regions of Granada, Menorca, and Ribera D’Ebre were started between 1997 and 2000, and the more recent ones in the regions of Asturias, Guipuzkoa, Sabadell, and Valencia were begun between 2004 and 2007. Exposure levels in the oldest cohorts are higher because the environmental levels of the chemicals were higher at that time. The concentrations of POPs in expectant mothers were measured using serum samples collected in pregnancy and cord blood samples collected at birth. Exposure to a wide list of other environmental pollutants was also measured by analyzing biological samples (blood, urine, hair) collected from the mothers during pregnancy. The heights and weights of the children from birth onward were taken from their medical records and also directly measured by the researchers at various ages. The researchers also used questionnaires to collect extensive data on demographics and lifestyle factors. Valvi first described the results from the newer cohorts, where the levels of exposure were lower (Mendez et al., 2011; Valvi et al., 2014). Both DDE exposure and hexachlorobenzene exposure were associated with the rapid growth of an infant in the first 6 months of life and a subsequent increase in the risk of being overweight at 1 year of age. Findings from these studies further suggested that the associations may be influenced by the child’s sex and, less definitively, by the maternal prepregnancy weight and the duration of exclusive breast-feeding. There was no evidence that prenatal exposure to PCBs was associated with either rapid growth or the likelihood of being overweight at age 1 year. Next Valvi described the results from the older Menorca birth cohort (Smink et al., 2008; Valvi et al., 2012). They found that prenatal exposure to hexachlorobenzene increased the risk for both being overweight and having obesity when the child was 7 years of age. There was also some suggestion of an association between prenatal exposure to DDE and being overweight at age 7 years, but the association was stronger for the second tertile than for the third tertile, that is, when the exposure levels were medium rather than high. Because the exposure levels were higher in the Menorca study, DDT was detectable in the vast majority of the cord blood samples analyzed; in contrast, in the later cohorts, most of the mothers had DDT concentrations below the limits of detection. Prenatal DDT exposure was nonlinearly associated with being overweight at age 7 years, but only in 

THE ROLE OF CHEMICAL EXPOSURES AND OBESITY 25 boys and in children who had higher levels of consumption of total fats in their diets and not in children with lower fat intakes. In contrast, prenatal exposure to PCBs was associated with a higher risk for being overweight at age 7 years in girls but not in boys. The INMA researchers further evaluated the associations between exposures to multiple chemicals, including 27 different endocrine disrupters whose levels were measured in maternal biological samples collected in pregnancy, and child BMI at age 7 years using principal component analyses. The findings from this multipollutant approach showed that associations between POPs and childhood obesity remain robust after accounting in the models for exposure to other chemicals thought to be linked to childhood obesity, including BPA (Valvi et al., 2013), phthalates (Valvi et al., 2015), and polybrominated diphenyl ethers (PBDEs) and metals (Agay-Shay et al., 2015). Summarizing the INMA birth cohort studies, Valvi said that exposures to DDE and hexachlorobenzene were associated with obesity- related outcomes both very early in life, in the first year of age, and later, at the age of 7 years. Exposure to PCBs was not linked to growth outcomes early in life, but there was a suggestion that prenatal exposure may increase weight only in girls, manifesting around the age of 7 years. For DDT there was a nonlinear association between exposure and weight, but only in boys and in children with high fat intakes. There was little evidence that these associations may be influenced by other potential modifiers: maternal prepregnancy weight and/or exclusive breast-feeding duration. Children’s Health and the Environment in the Faroes Study Valvi also discussed findings from the birth cohort studies in the Faroe Islands, which are situated between the Norwegian Sea and the northern Atlantic Ocean.4 The prevalence of overweight in Faroese children is much lower than that in Spanish children: just 22 percent of 5- to 7-year-olds but 37 percent of Spanish 6-year-olds were classified as overweight by use of the 2007 World Health Organization growth reference (WHO, 2007). The population of the Faroe Islands is very homogeneous in terms of demographics and lifestyle characteristics, Valvi said. Most of the exposure to POPs is due to the consumption of whale meat, because these islands are inhabited by whale hunters. 4 For more information, see http://www.chef-project.dk. 

26 ENVIRONMENTAL CHEMICAL EXPOSURES AND OBESITY Since 1986, five birth cohorts have been recruited and are being followed up periodically. Valvi showed results from the third cohort, which was recruited at about the same time that the INMA Menorca cohort was recruited. The researchers collected blood samples from the mothers during pregnancy and then from the children at 5, 7, and 13 years of age. They also measured the children’s weights and heights at the same ages. An advantage of this study is that exposure to POPs as well as other environmental contaminants was measured both prenatally and postnatally by the use of serum samples collected from mothers and children. The study found some evidence that prenatal exposure to POPs is associated with an increased risk of obesity at 7 years of age (Tang- Péronard et al., 2014). However, following up on these findings Valvi examined the growth of the children between the ages of 7 and 13 years and found that the children who were more highly exposed to both DDE and PCBs during pregnancy had a reduced BMI gain compared with the less exposed children; there was no such association for the serum concentrations of POPs of the children at 5 years of age, and no clear association with the risk for being overweight at 13 years of age was shown for either prenatal or postnatal exposures. Valvi speculated that the negative associations between prenatal exposures and gain in BMI could be due to puberty status, because at age 13 years some of the children had already entered puberty, while others had not. Furthermore, there is some evidence from other studies that prenatal exposure to chemicals may influence the time of puberty. One interesting finding of this study, Valvi said, is that prenatal exposure to these chemicals may be more critical than postnatal exposure. Conclusions of Valvi’s Presentation Finally, Valvi showed a couple of slides that summarized the current state of the evidence concerning prenatal exposure to POPs and obesity measures, mainly the BMI of the child. She drew several conclusions from these studies and the literature review: • Low-level exposure to POPs is associated with childhood growth and obesity. • Studies evaluating the persistence of the associations later in adolescence and adult life are currently lacking. 

THE ROLE OF CHEMICAL EXPOSURES AND OBESITY 27 • The prenatal period (and perhaps the early postnatal period, as prenatal and postnatal exposures in the first years of life are highly correlated and hard to disentangle) may be the most critical window of exposure. • Susceptibility may vary according to sex and perhaps also according to breast-feeding duration, maternal weight before pregnancy, and the child’s consumption of fat. Future research, Valvi said, will focus on continuation of the cohorts’ follow-up, evaluation of the persistence of the associations at later ages, and identification of windows of exposure and susceptibility. It is also important to improve the assessments of obesity and use more direct adiposity measurements than just BMI. More work is also needed to identify the most susceptible groups and to evaluate the overall obesogenic effects of mixtures of various chemicals and not just POPs. Finally, she said, further research is needed to better elucidate the mechanistic pathways by which these chemicals may influence growth. A way to do this is to integrate biomarker data into the human studies. This is not that simple, she said, because the most relevant biomarkers to be measured are not yet known. “But it is something that we are working on,” she said, “and the collaboration between experimental and epidemiological studies will help to elucidate which is the best way to study mechanistic effects.” Discussion Linda Birnbaum of NIEHS asked Valvi if her studies had examined exposures to dioxin-like PCBs, because mechanistic studies suggest that these can affect adipocyte growth and differentiation. Valvi answered that they have data on aryl hydrocarbon receptor activity but have not yet finalized the analysis. She also suggested that the effects of dioxin-like compounds may be more complex than what people may think because they act through a number of receptors, not just the aryl hydrocarbon receptor, which has so far been the main focus in human studies. Birnbaum also commented that she appreciated Valvi’s discussion of how the effects of exposure to POPs may differ between the sexes. This has too often been ignored in the past, she said. Valvi agreed and added that the effective study of such differences in the effects between sex will require larger studies that are costly and wider collaboration among existing cohorts. 

28 ENVIRONMENTAL CHEMICAL EXPOSURES AND OBESITY Sheela Sathyanarayana from the University of Washington asked Valvi how she handled the fact that the results of these sorts of epidemiological exposure studies can be confounded by the children’s diets, particularly their fat intake. The problem of such confounding is a major issue, Valvi acknowledged. Because diet is the main source of exposure to these chemicals, it is difficult to know which part of the effect is due to the chemicals and which part is due to high fat intake. Therefore, she said, in their studies she and her colleagues have accounted for dietary factors, such as fat or carbohydrate intakes. Estimates of the effect of the associations with prenatal exposures do not usually change when such dietary factors are taken into account, she said, but she indicated that they assessed the diet using food frequency questionnaires, which is not the most accurate method to use for dietary assessment. ENDOCRINE-DISRUPTING CHEMICALS, ONSET OF PUBERTY, AND OBESITY The second speaker was Frank Biro, the director of research in adolescent and transition medicine at the Cincinnati Children’s Hospital Medical Center and a professor in the Department of Pediatrics at the University of Cincinnati. He spoke about the effects of endocrine- disrupting chemicals on the onset of puberty and obesity. Puberty Biro began by defining puberty. It is a series of interrelated changes involving just about every system in the human body. There is a pubertal growth spurt that is the only time in postnatal life that there is an acceleration in the rate of growth. There are profound changes in body composition. There is a maturation of the adrenal axis and a reactivation of the hypothalamic-pituitary-gonadal axis, which is fully functioning in a full-term infant but which gets turned off in the first 6 months of life. There is achievement of the ability to reproduce. Puberty can be considered a window of susceptibility in two ways. First, it can be a sensitive window to environmental exposures, so the timing of puberty is affected by what is going on outside the body. Second, puberty can serve as a special window of susceptibility to later adult morbidity and mortality, such as breast cancer. Several physiological changes are associated with puberty. Adrenarche is the activation of the adrenal cortex for the production of adrenal 

THE ROLE OF CHEMICAL EXPOSURES AND OBESITY 29 androgens. Pubarche is the appearance of pubic hair. Thelarche is the appearance of breast tissue. Gonadarche is the appearance of secondary sexual characteristics and has traditionally been defined to be the gonadal production of sex steroids. Menarche is the age of the first menstrual period. Biro then described the sequence of events associated with puberty in girls determined from data from the Growth and Health Study carried out by the National Heart, Lung, and Blood Institute in the 1980s. It begins with adrenarche, the appearance of adrenal hormones from the adrenal gland. The first sign is an increase in ovarian volume, which is something that clinicians cannot see. With this comes the beginning of the pubertal growth spurt, as the girl begins to shoot up in height. After that comes the appearance of breast tissue, which is typically what clinicians use to define the onset of puberty, and the appearance of pubic hair. A year and a half or so after the appearance of breast tissue is the peak growth rate, and 6 to 12 months after that is the first menstrual period. The completion of puberty occurs with full development of the breasts and pubic hair, but a bit of growth still occurs even after the attainment of what appears to be full pubertal maturation. Relationship Between Obesity and Puberty A number of studies have reported an association between obesity and earlier breast maturation in girls. A recent study by Biro and colleagues (Biro et al., 2013) carried out with a group of 1,239 girls determined that BMI accounted for 14 percent of the variance in the age of onset of pubertal maturation, while race and ethnicity accounted for only 4 percent. This was the first study to find BMI to be a more important contributor to the onset of puberty than race and ethnicity, Biro said. In 1974, Frisch and McArthur proposed that puberty would start a year or two after a child reached a certain critical level of body fat (Frisch and McArthur, 1974). Their work would be validated much later. In 1997, Matkovic and colleagues pointed out that the relationship was actually between leptin, a hormone produced by fat cells, and earlier menarche. They reported that for every increase in the serum leptin concentration of 1 nanogram per milliliter, the age of menarche dropped by 1 month (Matkovic et al., 1997). In 2002, Grumbach argued that the studies with leptin clearly showed that leptin is necessary for kids to go into puberty but that it is 

30 ENVIRONMENTAL CHEMICAL EXPOSURES AND OBESITY not sufficient and that there must be an additional mechanism. That mechanism was proposed to be the gonadotropin-releasing hormone pulse generator, a group of neurons located near the hypothalamus (Grumbach, 2002). In 2012, Bianco (Biro, 2015) reported that increased adiposity was associated with the earlier activation of the luteinizing hormone pulse generator. This, Biro said, could be considered the gonadostat, if you will. However, potential mechanisms in addition to obesity impact the onset of breast development in girls, Biro said. He showed a graph that plotted the percentage of girls who have started breast development by a certain age (see Figure 3-1). For this graph, only the development of white girls was plotted. The solid blue line describes data published in 1997 by Herman- Giddens and colleagues (Herman-Giddens et al., 1997). At the time many researchers doubted their results because a significant percentage of girls were starting breast development much earlier than expected— about 5 percent by the age of 7 years, for instance. But, Biro said, later studies confirmed their data indicating the onset of breast development in girls earlier than had previously been thought. Then, in late 2013 Biro and colleagues published the results of a similar analysis done with girls who participated in the Breast Cancer and Environmental Research Program (BCERP). He showed curves representing the onset of breast development in white girls in that study (see Figure 3-1). Not surprisingly, breast development in the overweight and obese girls—those whose BMIs were above the 85th percentile— came much earlier than that in the girls that Herman-Giddens and colleagues had reported on. However, the girls whose BMIs were below the 85th percentile and whose average BMI was very similar to that of the girls in the study of Herman-Giddens and colleagues also showed an earlier onset of breast development: an average of about 8 months earlier than had been the case in 1997. What happened between 1997 and 2013? Biro and his colleagues have proposed that the earlier age of onset of breast development may be due to some environmental exposures that they are now evaluating. The relationship between adiposity and the onset of puberty in boys is a little bit less clear-cut, Biro said. Of more than a dozen papers that have examined this issue, several report that a higher BMI leads to an earlier onset of puberty, several note that a higher BMI leads to a later onset of pubertal maturation, and a few state that the relationship is not 

THE ROLE OF CHEM EMICAL EXPO OSURES AND O OBESITY 31 FIGUR RE 3-1 Onset of o breast development. NOTE: BCERC = Brreast Cancer an nd the Environmment Researchh Centers; BMII = body mass m index. SOURC CES: Biro preesentation to workshop, w Marrch 2, 2015, ffrom Biro et aal., 2013. Reproduced R wiith permission from Pediatriccs, vol. 132, pages 1019–10227, copyrig ght 2013 by thee American Accademy of Peddiatrics. clear. Typically, T he said, the Euroopean studies suggest that a higher BMII is associated with pub berty arrivingg a little bit eearlier, whilee the American studiess suggest thatt a higher BM MI is associaated with pubberty arrivingg a little bit b later. Biiro suggested that what maay be happeniing is that theere is a J-shapped or U-sshaped curvee that describ bes the relattionship betw ween BMI and puberttal onset in boys. b That is, an increasinng BMI leadds to an earliier onset of puberty until u the BMII reaches obeesity levels, as which poiint there is a delay in thhe onset of puubertal maturaation. Pu utting the various studiess together, B Biro suggested how varioous mechaanisms could play a role in the relatioonship betweeen BMI or tthe amoun nt of body fat and the onsett of puberty. H He noted thatt his model w was adapteed from a 20 008 paper by y Ahmed andd colleagues (Ahmed et aal., 2008). In the modell, various expo osures could llead to increaased obesity aand increassed amounts of visceral fat, f includingg exposures cconsisting off a consisttent energy im mbalance, en ndocrine-disrupupting chemiccals, and inadde- 

32 ENVIRONMENTAL CHEMICAL EXPOSURES AND OBESITY quate prenatal growth. As Biro noted, it is the babies who are small for their gestational age who have a higher propensity toward obesity. The larger amount of fat has various consequences: higher levels of leptin; increased levels of aromatase, which is the enzyme that converts androgens into estrogens; and insulin resistance and elevated insulin levels. The insulin resistance and elevated insulin levels in turn act directly on the adrenals and increase the production of the adrenal androgens, which leads to earlier adrenarche. Insulin also acts on the liver to lower the levels of sex hormone-binding globulin, which means that there is a greater bioavailability of the sex steroids. Elevated levels of insulin also act on the ovaries and lead to increased androgen production. After this, Biro spoke about some recent results showing an intriguing relationship between obesity and puberty. Using a sensitive method to measure serum estradiol levels, he and his colleagues examined those levels in a group of girls at various points in time as the girls approached and entered puberty. For the girls in the study who had a BMI below the median, the change in estradiol levels was as expected: a slow increase as they approached puberty and then a sharp increase as they entered puberty. But for the girls with a BMI above the median— and these were mostly overweight or obese girls—the estradiol levels barely increased as they went through pubertal maturation. At first, Biro said, he thought the results could not be true. How could these overweight and obese girls even be going through puberty? Then he recalled that obese women have higher rates of breast cancer and that the mechanism that has been proposed to explain that is that in these women’s fat cells, aromatase is converting adrenal androgens into estrogen, leading to higher levels of estrogens and an increased risk of breast cancer. So, Biro said, what he believes that this study shows is that while the overweight and obese girls are not getting a big increase in estradiol levels, they do have high local levels of estrogen because of the conversion of adrenal androgens in their fat cells without elevated serum estrogen levels. Thus, they are able to go through puberty but without the extra estradiol. Environmental Influences on Puberty Biro then described the study on the environmental and genetic determinants of puberty, BCERP. The study began 13 or 14 years ago with the goal of collecting markers of breast development and other 

THE ROLE OF CHEMICAL EXPOSURES AND OBESITY 33 physiological changes of sexual maturation to look at environmental stressors and see if they might be leading to future breast cancer risks. The stressors included lifestyle, nutrition, body size, and exposures. There were three sites for the BCERP: in East Harlem in New York City through the Mount Sinai School of Medicine, in the Bay Area of California through Kaiser Permanente of Northern California, and schools in the greater Cincinnati, Ohio, area supplemented with the daughters and granddaughters of women enrolled in the Breast Cancer Registry of Greater Cincinnati. The sites recruited girls from 6 to 8 years old and saw them yearly or every 6 months. Over 2.5 years they recruited more than 1,200 girls about evenly divided among black non-Hispanic, white non-Hispanic, and Hispanic girls. They were looking for the effects of endocrine-disrupting chemicals (EDCs), which interfere with how hormones are synthesized, how they are broken down, or how they act on the hormone receptor, sometimes by increasing the signal and sometimes by decreasing it. The specific effect of one of these chemicals may depend on the timing of exposure, Biro said. For example, one study found that soy formula consumption during infancy leads to earlier menarche, while two others found that soy consumption in childhood led to a delay in the onset of breast tissue development. The timing may be the critical piece, he said. EDCs have been found to act through a variety of molecular mechanisms. For example, BPA has been associated with increased aromatase activity, while phthalates and perfluorinated chemicals have been associated with decreased activities of 3β-hydroxysteroid dehydrogenase and 17β-hydroxysteroid dehydrogenase; 3β-hydroxysteroid dehydrogenase is involved in converting estrone to estradiol and androstenediol to testosterone, and 17β-hydroxysteroid dehydrogenase is associated with the production of the sex steroids. In animal experiments, it has been shown that BPA stimulates gene transcription for kisspeptin 1 (KiSS1), which has been proposed to be the hormone leading to the onset of puberty. The studies with the girls participating in BCERP did indeed find a number of environmental influences on puberty (Wolff et al., 2014). Biro first spoke about the results of studies with phthalates. Phthalates fall into two general categories, he explained: low-molecular-weight phthalates, which are found in fragrances and personal care products, and high- molecular-weight phthalates, which appear in soft plastics, sealants, and flooring. The researchers found an earlier onset of breast development in 

34 ENV VIRONMENTA AL CHEMICAL L EXPOSURESS AND OBESIITY girls exposed e to low-molecularr-weight phthhalates, but w with additionnal analysiis they found that the effectt seemed to wwork via an inccrease in BMI: girls exxposed to the phthalates haad higher averrage BMIs, w which in turn lled to an earlier onset of puberty. In I contrast, eexposure to hhigh-moleculaar- weightt phthalates was w associateed with a lateer appearancee of pubic haair, but thee effect was greater g in normmal-weight ggirls than in ovverweight girrls, whosee greater BMI may have haad a mediatingg effect (see F Figure 3-2). Thhe group has also looked at a the effects of phenols, which are ussed for a variety of applications: a antiseptics, ssunscreen, mmothballs, hannd sanitizzers, and morre. The chem mistry of the pphenols is sim milar to that of sex hoormones. For example, onee of the phen ols used in suunscreen, BPA, has alsso been used d in the past asa a pharmaccological estrrogen, so it hhas estrogeenic propertiees. According g to a study wwhose results Biro showed at the wo orkshop but which w has noot been publiished, variouus phenols haave been liinked with efffects on breasst developmeent. Both the ssunscreen ageent benzop phenone-3 annd enterolactoone were assoociated with laater maturatioon, while both 2,5-dicchlorophenol,, a chemicall found in m mothballs, annd triclosan led to an earlier e onset of o breast deveelopment. FIGUR RE 3-2 Delay yed appearancce of pubic haair in girls exxposed to higgh- molecuular-weight phtthalates. NOTE: DEHP = bis(2 2-ethylhexyl) phthalate; p gC = creatinine. SOURC CES: Biro preesentation to workshop, w Marrch 2, 2015, frrom Wolff et aal., 2014. 

THE ROLE OF CHEMICAL EXPOSURES AND OBESITY 35 Like phenols, perfluorinated chemicals have widespread applications in consumer products, such as in grease repellents and water stain repellents, and also industrial applications, such as in the production of Teflon, and research has shown that exposure to these chemicals is widespread in the population. Studies have shown various effects, including exposure to perfluorooctanoic acid being linked with shifts in the onset of breast development, especially among normal-weight girls. In conclusion, Biro said that it will be important to examine the effects of exposure to some of the replacement chemicals now being used, such as bisphenol S, which is being used in place of BPA in a number of applications. It will also be important to begin looking at the effects of mixtures of compounds and not just single chemicals. “For better or worse, we live in an ocean of compounds,” he said. “It is much more difficult to try to sort through what is happening with a mixture of compounds.” In the brief question-and-answer period after the presentation, an audience participant asked if Biro and his colleagues had considered alternative approaches to modeling, such as structural equation modeling, to disentangle the effects of the multiple chemicals that people are exposed to. Biro responded that he and his colleagues have collected a vast amount of data on the girls in their studies: dietary patterns, anthropometric examinations, urine and serum biomarkers, sex steroids, fasting insulin, and glucose, among others. “Right now, we are dealing with two and three parameters at a time,” he said. “After we have sorted through those, then we will start looking at these much more complicated models…. I think that after we control for some of these other mechanisms and start exploring how these mechanisms are related to each other, we will start getting a better story about these exposures.” OBELIX The third presenter was Juliette Legler, a professor of toxicology and environmental health and the deputy head of the Department of Chemistry and Biology in the Institute of Environmental Studies at the Vrije University Amsterdam in the Netherlands. She was the director of the OBELIX project, which studied possible links between early life exposure to endocrine-disrupting chemicals and the development of obesity later in life. OBELIX, Legler explained, was named for a famous French cartoon character, a very large, strong man who got his strength and his huge size 

36 ENVIRONMENTAL CHEMICAL EXPOSURES AND OBESITY as a young boy by falling into a cauldron of magic potion. The question is, What was in that cauldron? That, in a sense, is what OBELIX is trying to find out for real people. The project involved seven institutes in five European countries. It lasted 4.5 years and finished at the end of 2013. At the time of the workshop, Legler said, researchers were still busy getting all the resulting papers published and working on a large integrated review paper that was to be submitted soon. The basic hypothesis behind OBELIX, Legler said, was that perinatal exposure to EDCs plays a role in the development of obesity later in life, and the project examined various mechanisms that could explain that connection. EDCs could, for example, cause changes in adipocyte differentiation. They could also cause a change in birth weight that would lead to changes in a child’s long-term growth trajectories. They could affect early growth and BMI in children, or they might cause changes in hormone and lipid metabolism. OBELIX involved a collaboration between epidemiologists and toxicologists who informed each other’s work. The epidemiologists carried out studies on four mother-and-child cohorts from Belgium, the Netherlands, Norway, and Slovakia, while the toxicologists performed animal studies and in vitro mechanistic studies. The epidemiologists and toxicologists studied four classes of compounds in common: non-dioxin-like PCBs, perfluorinated compounds, dioxin-like compounds, and phthalates. The epidemiologists also carried out studies on brominated flame retardants and organochlorine pesticides, while the toxicologists carried out an additional study on BPA. “We selected these compounds because we were very interested in different types of endocrine-disrupting chemicals,” Legler explained. “It was a fact- finding mission looking at different endocrine mechanisms that they could disrupt and if this could perhaps be linked to the potential obesogenic effects of these compounds.” The epidemiologists carrying out the studies determined exposures to the various compounds by examining cord blood or the mother’s milk, or both. The children were up to 6 or 7 years of age at follow-up. The OBELIX epidemiologists worked with researchers across Europe who were carrying out studies on their own birth cohorts to expand the number and breadth of studies. In one of the first such collaborative studies, the researchers performed a detailed exposure assessment of PCB and DDE levels in cord blood in approximately 8,000 children in various cohorts throughout Europe. When they compared 

THE ROLE OF CHEMICAL EXPOSURES AND OBESITY 37 exposure to birth weight, they found that PCB exposure was significantly associated with a decreased birth weight (Govarts et al., 2012). It was one of the largest studies of its kind indicating an association between PCBs and lower birth weight, Legler said. The OBELIX team wanted to know if this lower birth weight would translate into changes in growth and weight in these children as they got older, she said. So they followed the children in their studies up to 7 years of age and measured growth, BMI, and the levels of certain metabolic hormones, such as leptin, adiponectin, and insulin. The complementary animal studies were designed to be very similar in design to the human studies. The mice were exposed to environmental chemicals through their mothers’ blood supply during gestation and for another 3 weeks after birth through breast-feeding until the animals were weaned. The mice were given one of eight different doses, all of which were below the level at which developmental toxicities are observed. In other words, Legler said, they were low, nontoxic doses. The animals then grew to adulthood, and some of them were given a high-fat diet. The various endpoints measured included body weight; fat pad weight; histopathology; food consumption; physical activity; serum lipid and insulin levels; as well as leptin, adiponectin, and glucagon levels. Legler provided a brief look at the results of the OBELIX studies, many of which are still under review at different journals. In one case, for instance, there was a significant increase in growth in early life related to perinatal exposure to dioxin-like compounds, with exposed children being about 350 grams heavier at 2 years of age than unexposed children. However, when the children were followed to 7 years of age, there was a positive association only in girls and not in boys. A separate study with a smaller cohort looked at the levels of leptin, adiponectin, and insulin in the blood and found a significant negative association between exposure and levels of serum adiponectin in both boys and girls. Serum adiponectin, Legler noted, is an important hormone involved in regulating glucose levels and fatty acid breakdown. In the related animal studies in which mice were exposed to dioxin- like compounds before and after birth, after 1 year the exposed female mice were significantly heavier than the unexposed female mice, particularly when they were given a high-fat diet. And not only were the mice heavier, but they also developed more fat tissue. In studies with perfluorooctanoic acid (PFOA), exposed children grew more quickly up to 24 months of age, but there was no difference in growth between exposed and unexposed children at 7 years of age. In the 

38 ENVIRONMENTAL CHEMICAL EXPOSURES AND OBESITY animal studies, there was actually a decrease in weight among exposed female mice. Thus, in contrast to the dioxin-like chemicals, which had an obesogenic effect, PFOA did the opposite in females and led to weight loss. In their in vitro studies the OBELIX researchers studied, among other things, the effects of EDCs on the differentiation of preadipocytes into mature fat cells. Some EDCs, such as tributyltin and brominated diphenyl ether 47 (BDE 47), a flame retardant, increased differentiation. However, other chemicals, such as tetrachlorodibenzodioxin (TCDD), actually inhibited fat cell differentiation. The researchers also found dose–response relationships for some of the chemicals, with the amount of adipocyte differentiation increasing as the dose increased. This is strong evidence that some of these compounds—including tributyltin, BPA, and BDE 47—may induce fat cell differentiation. Further investigation showed that several of these chemicals affected global DNA methylation in fat cells. DNA methylation, the biochemical process of adding methyl groups to certain sites on a DNA molecule, can reduce the expression of a gene to which the methyl group has been attached. The researchers found that some chemicals, such as tributyltin, led to decreased methylation, while others, such as BPA, increased the level of methylation. “This gave us some indication that … the stimulation of adipocyte differentiation is accompanied by changes in DNA methy- lation,” Legler said. “We were interested to know what genes were involved here.” One of the genes involved, she said, is PPARγ2, the so-called master regulator of fat cell differentiation. The researchers showed that BDE 47 causes a decrease in the methylation of the promoter sequence of the PPARγ2 gene, which leads to increased expression of the gene and increased fat cell differentiation, so this appears to be the mechanism by which BDE 47 leads to an increased number of fat cells. In conclusion, Legler said that the OBELIX project provided evidence that endocrine-disrupting chemicals do indeed play a role in obesity, affecting growth and metabolic pathways. Not all of the chemicals led to heavier phenotypes, she noted. Some of them did, but some of them actually led to leaner phenotypes. In particular, there was evidence from both epidemiological and animal studies that DDE and dioxin-like compounds lead to heavier phenotypes, while there was evidence that PCB 153 inhibits growth in children. “We do believe that the current levels of EDCs may pose a risk for metabolic disruption,” Legler said, but the effects vary by chemical, by 

THE ROLE OF CHEMICAL EXPOSURES AND OBESITY 39 sex, and by time of exposure. There are clear differences between the effects on males and those on females and between prenatal and postnatal effects. It will be important, she added, to see if these short-term effects on adiposity and BMI have long-term consequences. She said that it is known that obesity in early childhood is a predictor of long-term obesity, so it seems likely that there will indeed be long-term effects, but the data are lacking. “We really would like to follow up with these kids much longer,” she said. In the future, she said, her lab will be following up on the OBELIX cohorts as part of another European project that is focused on neuro- development. In particular, researchers in her lab have been working for the past 3 or 4 years on an alternative model of obesity using the zebrafish model. One of the advantages of using zebrafish is that they are transparent when young. It is possible to stain a young fish with a lipid stain that causes fat cells to stand out and then watch as the fat tissue develops. The research on the zebrafish has shown that certain environmental chemicals, such as flame retardants and UV filters, disturb both the metabolism and the circadian rhythms in the exposed fish. The fish “show absolutely no circadian rhythm anymore when they are exposed to the UV filter,” Legler said. “We are really wondering what is the chicken and what is the egg. Are these chemicals affecting circadian rhythm and that is affecting metabolism, or the other way around? Is metabolism affected by obesogenic chemicals that disrupt circadian cycling?” The research may eventually point to alternative mechanisms by which chemical exposures can affect metabolism, she said. DISCUSSION In the general discussion session following the three presentations described above, Kristina Rother from the National Institutes of Health asked whether the various epidemiological studies discussed in the session had taken socioeconomic status into account. Legler answered that the epidemiologist on her team did see differences according to socioeconomic status and used socioeconomic classes as a covariate in the models. In particular, in the Slovakian cohort, about two-thirds of the children were of Roma origin. Legler noted that the greatest effects on obesity as well as the highest exposure levels were in this class and that it was certainly an important factor included in the analyses. 

40 ENVIRONMENTAL CHEMICAL EXPOSURES AND OBESITY Biro added that not only socioeconomic status but also race/ethnicity and the specific environments in which people live must be taken into account. All these factors are interrelated, and it can be difficult to tease apart their influences. Rother suggested that one connection between obesity and circadian rhythms is that the effect of a particular chemical may vary according to when in a circadian cycle one is exposed. Insulin is known to have a circadian cycle, for instance, with insulin responses to eating being higher in the morning than in the evening. So perhaps the body’s responses to obesogens might vary by time of day as well. Legler responded that her group has not looked at that particular issue and that, indeed, the animals are exposed to chemicals continuously through the 24-hour cycle. This may have masked a specific daytime- or nighttime- related effect, she speculated. She also noted that circadian rhythms do not appear until a certain point in development when the brain is far enough along in development that the circadian cycle is established. Thus, whether a chemical affects the development of the circadian rhythm would depend on the timing of the exposure. Judy LaKind from the University of Maryland School of Medicine, who was watching via the webcast, asked if the models for postnatal exposure and BMI took into account the fact that breast-fed children often have a growth rate lower than that of formula-fed children. Legler responded that in the European studies there were essentially no women who did not breast-feed, so the only related factor that could be examined was the amount of time that the children were breast-fed. Legler added that there was a close relationship between the amount of prenatal exposure to a particular chemical and the amount of postnatal exposure through breast-feeding, so it was impossible to completely isolate the effects of the two types of exposures. “But I think the important point we wanted to make here,” she said, “is you cannot neglect how important postnatal exposure is for some of these exposures, certainly for women who have a longer breast-feeding duration.” Valvi added that in her studies she has included detailed information on the duration of breast-feeding and also on exclusive breast-feeding that further accounted for the age of introduction of solid foods and formula milk and that when she adjusted for the duration of breast- feeding, the associations were attenuated but remained significant. Sandra Haslam of Michigan State University asked Legler whether she fed some of the animals in her study a diet of regular chow, in addition to giving some animals a high-fat diet. Then she asked whether 

THE ROLE OF CHEMICAL EXPOSURES AND OBESITY 41 Legler’s group had taken into account the differences in diet among the subjects of their studies. Legler answered that in most cases she saw no significant effects on body weight in animals fed a normal chow diet, which is why in most of the studies the animals were given a high-fat challenge at the end of the follow-up period. The one exception was that BPA-exposed male mice on the normal chow diet did show a gain in body weight beginning in early development. In humans, she said, she and her colleagues found it difficult to get reliable information on the mothers’ diets when they were pregnant and breast-feeding, and they saw no correlation between a mother’s diet and her child’s weight in the future. Sally Darney from the U.S. Environmental Protection Agency asked if anyone had looked at the possible effects of geographical location on obesity. She pointed out that the data that Valvi presented indicated a higher prevalence of obesity in the southern European countries and that the southeastern United States also has a higher rate of obesity. So could latitude make a difference? Could growing up in a warm climate instead of a cold one have long-term effects on weight? Valvi said that she does not think that there is “some weird confounding” related to latitude that affects the relationships between environmental chemical exposures and obesity, because pooled analysis in recent collaborative European projects have shown associations to be in the same direction across cohorts. Furthermore, she said, she thought that genetic differences or perhaps differences in diet or physical activity between the northern and the southern European countries was a more likely explanation for the differences in obesity prevalence than latitude. Still, she said, there are data indicating that climate conditions influence birth weight, which is an important determinant for obesity later in life. Thus, she said, it might also be that there is a relationship between climate or geographical latitude and obesity. Archana Lamichhane from the University of North Carolina at Chapel Hill asked about what effects a mother’s gestational diabetes might have on her child’s birth weight and later risks of obesity and other metabolic diseases. Valvi responded that a number of studies indicate that prenatal exposure to various environmental chemicals may increase the child’s risk for diabetes later in life. “And if prenatal exposure can increase the risk during postnatal life,” she said, “why would not exposing the mother increase the risk for diabetes appearing during pregnancy? That is a possible scenario.” Thus, she continued, it is possible that gestational 

42 ENVIRONMENTAL CHEMICAL EXPOSURES AND OBESITY diabetes mediates some of the effects that they see between prenatal chemical exposures and childhood obesity. It is also possible that gestational diabetes serves as a confounder because diabetes during pregnancy could change the metabolism of chemicals in the mothers and may explain the associations with child obesity shown later in life. Either way, she concluded, it is clearly an important factor to consider. Sangeeta Khare of the U.S. Food and Drug Administration asked if the method of delivery—naturally versus by cesarean section—has any effects on obesity. Valvi responded that the type of delivery has been associated with both birth weight and later metabolic risk. However, controlling for the type of delivery in the models was not shown to influence the associations. Legler said that it is possible that children delivered by cesarean section may be exposed to certain chemicals, such as BPA, because of the medical care associated with such a delivery. In particular, she said, premature babies are generally attached to various sorts of tubes—feeding tubes, breathing tubes, and so on—and that could result in an exposure to certain chemicals in the period just after birth. Valvi then commented that their studies would probably not have captured exposures to the variety of nonpersistent chemicals that are used as part of medical practice and care at the time of or immediately after birth. REFERENCES Agay-Shay, K., D. Martinez, D. Valvi, R. Garcia-Esteban, X. Basagaña, O. Robinson, M. Casas, J. Sunyer, and M. Vrijheid. 2015. Exposure to endocrine-disrupting chemicals during pregnancy and weight at age 7 years of age: A multi-pollutant approach. Environmental Health Perspectives. DOI:10.1289/ehp.1409049. Ahmed, M. L., K. K. Ong, and D. B. Dunger. 2008. Childhood obesity and the timing of puberty. Trends in Endocrinology and Metabolism 20(5):237–242. Biro, F. 2015. The interplay between environmental exposures and obesity: Onset of puberty, obesity, and endocrine disrupting chemicals. Presentation to the Roundtable on Environmental Health Sciences, Research, and Medicine. Biro, F. M., L. C. Greenspan, M. P. Galvez, S. M. Pinney, S. Tieitelbaum, G. C. Windham, J. Deardorff, R. L. Herrick, P. A. Succop, R. A. Hiatt, L. H. Kushi, and M. S. Wolff. 2013. Onset of breast development in a longitudinal cohort. Pediatrics 132(5):1–9. Frisch, R. E., and J. W. McArthur. 1974. Menstrual cycles: Fatness as a determinant of minimum weight for height necessary for their maintenance or onset. Science 185(4155):949–951. 

THE ROLE OF CHEMICAL EXPOSURES AND OBESITY 43 Govarts, E., M. Nieuwenhuijsen, G. Schoeters, F. Ballester, K. Bloemen, M. de Boer, C. Chevrier, M. Eggesbø, M. Guxens, U. Krämer, J. Legler, D. Martínez, L. Palkovicova, E. Patelarou, U. Ranft, A. Rautio, M. S. Petersen, R. Slama, H. Stigum, G. Toft, T. Trnovec, S. Vandentorren, P. Weihe, N. W. Kuperus, M. Wilhelm, J. Wittsiepe, J. P. Bonde; OBELIX; ENRIECO. 2012. Birth weight and prenatal exposure to polychlorinated biphenyls (PCBs) and dichlorodiphenyldichloroethylene (DDE): A meta-analysis within 12 European birth cohorts. Environmental Health Perspectives 120(2):162–170. Grumbach, M. M. 2002. The neuroendocrinology of human puberty revisited. Hormone Research 57(Suppl 2):2–14. Herman-Giddens, M., E. J. Slora, R. C. Wasserman, C. J. Bourdony, M. V. Bhapkar, G. G. Koch, and C. M. Hasemeier. 1997. Secondary sexual characteristics and menses in young girls seen in office practice: A study from the Pediatric Research in Office Settings Network. Pediatrics 99(4):505–512. La Merrill, M., E. Karey, E. Moshier, C. Lindtner, M. R. La Frano, J. W. Newman, and C. Buettner. 2014. Perinatal exposure to mice to the pesticide DDT impairs energy expenditure and metabolism in adult female offspring. PLoS ONE 9(7):e103337. Matkovic, V., J. Z. Ilich, N. E. Badenhop, M. Skugor, A. Clairmont, D. Klisovic, and J. D. Landoll. 1997. Gain in body fat is inversely related to the nocturnal rise in serum leptin level in young females. Journal of Clinical Endocrinology and Metabolism 82(5):1368–1372. Mendez, M. A., R. Garcia-Esteban, M. Guxens, M. Vrijheid, M. Kogevinas, F. Goñi, S. Fochs, and J. Sunyer. 2011 Prenatal organochlorine compound exposure, rapid weight gain, and overweight in in infancy. Environmental Health Perspectives 119(2):272–278. Smink, A., N. Ribas-Fito, R. Garcia, M. Torrent, M. A. Mendez, J. O. Grimalt, and J. Sunyer. 2008. Exposure to hexachlorobenzene during pregnancy increases the risk of overweight in children aged 6 years. Acta Pediatrica 97(10):1465–1469. Tang-Péronard, J. L., B. L. Heitmann, H. R. Andersen, U. Steuerwald, P. Grandjean, P. Weihe, and K. Jensen 2014. Association between prenatal polychlorinated biphenyl exposure and obesity development at ages 5 and 7 y: A prospective cohort study of 656 children from the Faroe Islands. American Journal of Clinical Nutrition 99(1):5–13. Valvi, D., M. A. Mendez, D. Martinez, J. O. Grimalt, M. Torrent, J. Sunyer, and M. Vrijheid. 2012. Prenatal concentrations of polychlorinated biphenyls, DDE, and DDT and overweight in children: A prospective birth cohort study. Environmental Health Perspectives 120(3):451–457. Valvi, D., M. Casas, M. A. Mendez, A. Ballesteros-Gómez, N. Luque, S. Rubio, J. Sunyer, and, M. Vrijheid. 2013. Prenatal bisphenol A urine concentrations and early rapid growth and overweight risk in the offspring. Epidemiology 24(6):791–799. 

44 ENVIRONMENTAL CHEMICAL EXPOSURES AND OBESITY Valvi, D., M. A. Mendez, R. Garcia-Esteban, F. Ballester, J. Ibarluzea, F. Goñi, J. O. Grimalt, S. Llop, L. S. Marina, E. Vizcaino, J. Sunyer, and M. Vrijheid. 2014. Prenatal exposure to persistent organic pollutants and rapid weight gain and overweight in infancy. Obesity 22(2):488–496. Valvi, D., M. Casas, D. Romaguera, N. Monfort, R. Ventura, D. Martinez, J. Sunyer, and M. Vrijheid. 2015. Prenatal phthalate exposure and childhood growth and blood pressure: Evidence from the Spanish INMA-Sabadell birth cohort study. Environmental Health Perspectives DOI:10.1289/ehp.1408887. WHO (World Health Organization). 2007. Growth reference data for 5–19 years. http://www.who.int/growthref/en (accessed March 23, 2016). Wolff, M. S., S. L. Teitelbaum, K. McGovern, G. C. Windham, S. M. Pinney, M. Galvez, A. M. Calafat, L. H. Kushi, F. M. Biro, and the Breast Cancer and Environment Research Program. 2014. Phthalate exposure and pubertal development in a longitudinal study of U.S. girls. Human Reproduction 29(7):1558–1566.