Endocrine disruption of oestrogen action and female reproductive tract cancers

The oestrogenic activities of DES has been comprehensively studied and is described

Abstract

Endocrine disrupting chemicals (EDC) are ubiquitous and persistent compounds that have the capacity to interfere with normal endocrine homoeostasis. The female reproductive tract is exquisitely sensitive to the action of sex steroids, and oestrogens play a key role in normal reproductive function. Malignancies of the female reproductive tract are the fourth most common cancer in women, with endometrial cancer accounting for most cases. Established risk factors for development of endometrial cancer include high BMI and exposure to oestrogens or synthetic compounds such as tamoxifen. Studies on cell and animal models have provided evidence that many EDC can bind oestrogen receptors and highlighted early life exposure as a window of risk for adverse lifelong effects on the reproductive system. The most robust evidence for a link between early life exposure to EDC and adverse reproductive health has come from studies on women who were exposed in utero to diethylstilbestrol. Demonstration that EDC can alter expression of members of the HOX gene cluster highlights one pathway that might be vulnerable to their actions. In summary, evidence for a direct link between EDC exposure and cancers of the reproductive system is currently incomplete. It will be challenging to attribute causality to any single EDC when exposure and development of malignancy may be separated by many years and influenced by lifestyle factors such as diet (a source of phytoestrogens) and adiposity. This review considers some of the evidence collected to date.

Diethylstilbestrol

DES, a synthetic non-steroidal oestrogen, is often regarded as the archetypal endocrine disruptor. From about 1940 to 1970, DES was given to pregnant women in the mistaken belief that it would reduce the risk of pregnancy complications. Herbst et al. reported a probable link between DES and vaginal clear cell adenocarcinoma in girls and young women who had been exposed to this drug. It is estimated that five to ten million people were exposed to DES, including the pregnant mothers who received treatment and their offspring.

Of the several million women exposed to DES in utero, a cohort of 4653 DES-exposed women have been followed up to investigate the long-term consequences of exposure. Patient data stratified to account for the extent of exposure or dose effects of DES identified an association between treatment of mothers earlier during their pregnancy and adverse vaginal epithelial changes at a younger age in their offspring. While DES exposure is associated with increased risk of breast and cervical/vaginal clear cell adenocarcinoma, several studies have indicated that there is no associated risk of endometrial or ovarian cancer. As endometrial cancer is most likely to present after menopause, many of the DES-exposed women may not yet be old enough to determine whether they are at excess risk, as in the 2011 report only 27% were older than 50 years. Although to date epidemiological data indicate that DES-exposed women may not be at increased risk of developing endometrial cancer, studies in animal models provide evidence to the contrary.

In the early 1990s, Newbold et al. developed a mouse model for investigating hormonal carcinogenesis in mice by investigating the effects of neonatal exposure to oestrogens on cancer development. Treatment of CD1 neonatal mice with DES on postnatal days 1–5, which correspond to late prenatal human development, resulted in 90% of DES-exposed mice developing uterine adenocarcinomas after 18 months while none of the control animals had neoplastic lesions. Crucially, while administration of DES increased the risk of uterine adenocarcinoma, endogenous oestrogen was required for tumour development with prepubertal ovariectomy preventing tumour development. In DES-exposed women, vaginal and cervical carcinomas were only detected post-menarche consistent with a requirement for endogenous oestrogen in tumour development. ERα knockout mice (ERKO) did not develop tumours following neonatal DES exposure; transgenic mice overexpressing ERα displayed accelerated tumour development but mice with a dominant negative isoform of ERα (ERΔ3) were not protected, highlighting the complexity of the molecular signalling mechanisms involved.

Interestingly, gene expression analysis indicates that developmental DES exposure results in persistent altered gene expression of oestrogen-responsive genes in the uterus that may explain the increased susceptibility to tumour development. Gene ontology analysis of microarray data revealed altered expression of genes involved in cell growth, differentiation and adhesion. Kabbarah et al.  collected uterine cancer tissue RNA from DES-exposed mice by laser capture microdissection to minimise contamination with other cell types and performed targeted transcriptional profiling. Interestingly, the tumour suppressor PTEN was down-regulated in the majority of tumours, analogous to loss of PTEN expression in human tumours. In addition, genes associated with cell adhesion, such as Decorin, were down-regulated in DES-induced tumours while suppressor of cytokine signalling 3 (Socs3) was over-expressed. Other studies have also identified molecular similarities between DES-induced tumours in mice and endometrial cancer in humans, such as microsatellite instability brought about by defects in expression of DNA mismatch repair genes such as MSH2 and MSH6.

It could be argued that the apparent trans-generational effect of endocrine disruption is of greater significance. Following neonatal DES exposure in mice, the F1 generation of DES daughters have an increased incidence of uterine adenocarcinoma. Newbold et al. found that 31% of F1 females from the maternal germ cell lineage developed tumours after 18 months despite there being no exogenous endocrine exposure in these animals, highlighting the potential for future risk to the daughters of DES-exposed women. DES is reported to induce epigenetic changes. Altered methylation patterns have been reported for several uterine genes that are permanently dysregulated after developmental DES exposure; lactoferrin and c-Fos are permanently up-regulated following neonatal DES exposure to due to hypomethylation of the promoter region.

DES has been reported to promote hypermethylation of the homeobox gene Hoxa10 in mice exposed in utero to DES. DES exposure was also associated with increased expression of DNA methyltransferases 1 and 3b leading to long-term altered expression of Hoxa10. Contrary to the reported action of DES on Hoxa10, exposure to BPA in mice in utero results in hypomethylation of the Hoxa10 promoter, which leads to enhanced binding of ERα to EREs in the promoter region and an increase in an ERE-driven reporter gene in vitro.

Thus, epigenetic changes in uterine genes may indicate a possible mechanism for trans-generational effects of DES because altered expression of genes is reported to persist in DES-lineage females.

References

  • Full study (free access) : Endocrine disruption of oestrogen action and female reproductive tract cancers, Endocrine-related cancer, doi.org/10.1530/ERC-13-0342, 2014 Mar.
  • Featured image credit alpco.
DES DIETHYLSTILBESTROL RESOURCES

Effect of DES exposure on fetal testis development and function

Human Reproduction Update, human experimental data systematic review, 2019

Abstracts

BACKGROUND

Overall, the incidence of male reproductive disorders has increased in recent decades. Testicular development during fetal life is crucial for subsequent male reproductive function. Non-genomic factors such as environmental chemicals, pharmaceuticals and lifestyle have been proposed to impact on human fetal testicular development resulting in subsequent effects on male reproductive health. Whilst experimental studies using animal models have provided support for this hypothesis, more recently a number of experimental studies using human tissues and cells have begun to translate these findings to determine direct human relevance.

OBJECTIVE AND RATIONALE

The objective of this systematic review was to provide a comprehensive description of the evidence for effects of prenatal exposure(s) on human fetal testis development and function. We present the effects of environmental, pharmaceutical and lifestyle factors in experimental systems involving exposure of human fetal testis tissues and cells. Comparison is made with existing epidemiological data primarily derived from a recent meta-analysis.

SEARCH METHODS

For identification of experimental studies, PubMed and EMBASE were searched for articles published in English between 01/01/1966 and 13/07/2018 using search terms including ‘endocrine disruptor’, ‘human’, ‘fetal’, ‘testis’, ‘germ cells’, ‘testosterone’ and related search terms. Abstracts were screened for selection of full-text articles for further interrogation. Epidemiological studies involving exposure to the same agents were extracted from a recent systematic review and meta-analysis. Additional studies were identified through screening of bibliographies of full-texts of articles identified through the initial searches.

OUTCOMES

A total of 25 experimental studies and 44 epidemiological studies were included. Consistent effects of analgesic and phthalate exposure on human fetal germ cell development are demonstrated in experimental models, correlating with evidence from epidemiological studies and animal models. Furthermore, analgesic-induced reduction in fetal testosterone production, which predisposes to the development of male reproductive disorders, has been reported in studies involving human tissues, which also supports data from animal and epidemiological studies. However, whilst reduced testosterone production has been demonstrated in animal studies following exposure(s) to a variety of environmental chemicals including phthalates and bisphenol A, these effects are not reproduced in experimental approaches using human fetal testis tissues.

WIDER IMPLICATIONS

Direct experimental evidence for effects of prenatal exposure(s) on human fetal testis development and function exists. However, for many exposures the data is limited. The increasing use of human-relevant models systems in which to determine the effects of environmental exposure(s) (including mixed exposures) on development and function of human tissues should form an important part of the process for assessment of such exposures by regulatory bodies to take account of animal–human differences in susceptibility.

Introduction

Development of the male reproductive system and its subsequent function is impacted by events that occur in utero. Perturbations in testicular development or function during fetal life may result in male reproductive disorders that present postnatally. This includes anatomical abnormalities identified at birth, such as cryptorchidism and hypospadias, or disorders presenting in adulthood, including testicular cancer or infertility. These associated disorders are collectively referred to as the testicular dysgenesis syndrome (TDS). The development of TDS has been shown in rats to be influenced by a reduction in androgen production or action during a key period of fetal life, known as the masculinization programming window (MPW). The increasing incidence of TDS disorders over recent decades, highlights the potential importance of environmental impacts in their etiology. Environmental factors that have been proposed to affect fetal testis development and predispose to TDS disorders include environmental chemicals (e.g. plasticizers and pesticides), pharmaceuticals (e.g. analgesics, metformin and diethylstilboestrol) and lifestyle factors (e.g. diet, alcohol and smoking).

Pharmaceuticals : Diethylstilboestrol

Diethylstilboestrol (DES) is a synthetic estrogen that was used clinically to prevent spontaneous miscarriage and pre-term labor from the 1940 s until the early 1970 s. DES was withdrawn from clinical use after the demonstration of a causal role in the development of vaginal carcinoma in girls born to exposed mothers. In addition to the effects on female offspring, an association with structural abnormalities of the male reproductive tract was also described including epididymal cysts, microphallus and testicular hypoplasia.

Animal studies

Animal studies involving in-vitro culture of rat and mouse fetal testis, have reported a reduction in testosterone production following exposure to DES, similar to the results of previous in-vitro studies involving fetal mice and in-vivo studies in rats.

Epidemiology

For TDS disorders, which are linked to a reduction in androgen action during fetal life, there is conflicting evidence regarding their association with maternal DES exposure. Three studies have reviewed the literature relating to exogenous estrogen exposure and male reproductive disorders. Whilst early studies reported that hypospadias was significantly associated with DES exposure, it has subsequently been pointed out that this related to urethral abnormalities resulting from exposure to exogenous estrogens (including DES), which may have resulted from abnormalities in penile development rather than an effect on urethral formation as a result of reduced androgen exposure. The meta-analysis of all available evidence revealed a significant association between DES exposure and hypospadias; however, it was concluded that any effect of DES on hypospadias is likely to be small. For cryptorchidism, an increased risk in association with DES exposure is reported; however, this was dependent on the statistical model used and was indicative of heterogeneity. A subsequent cohort study has reported an association between in-utero exposure to DES and an increased risk of cryptorchidism, however, only for those in whom the initial exposure occurred prior to the 11th week of gestation with no significant association following exposure after 11 GWs. Studies have demonstrated no effect of prenatal DES exposure on sperm counts or fertility; however, this is in contrast to a previous study demonstrating an association between prenatal exposure to DES and semen parameters in adult men. Importantly, this study included analysis of men born to a large cohort of mothers who participated in an RCT involving DES exposure during pregnancy.

Experimental evidence from human studies

To date, only two studies have investigated the effect of DES exposure on the human fetal testis (Table VI). In-vitro organ culture of first trimester human fetal testis exposed to DES (10−5 to 10−6 M) for 3 days did not alter testosterone production. Interestingly, this study compared effects of DES exposure in rodent and human fetal testis demonstrating contrasting results between species using an identical experimental system.

In a separate study using the xenograft model, exposure to DES (100μg/kg, three times weekly) for 35 days resulted in no significant difference in testosterone production by second trimester (15–19 GW) testis tissue. Interestingly, host mouse seminal vesicles were significantly increased in weight, which was indicative of increased testosterone production from the xenografted tissue over the entire grafting period. The reason for this unexpected increase in testosterone is unclear.

Summary

Whilst rodent studies have indicated a profoundly negative effect of DES exposure on testosterone production by the fetal testis, experimental studies utilizing human fetal testis tissues have failed to identify similar effects, which may relate to the presence of ESR1 in rodent Leydig cells, and the absence of this estrogen receptor in human fetal testis. Epidemiological data suggests that any potential effect of DES exposure on male reproductive development is likely to be of small magnitude. Taken together the results suggest an important species difference in terms of DES effects on fetal testosterone production which may explain why this frequently results in the development of male reproductive disorders in rodents, whilst associations between DES and subsequent male reproductive disorders in humans are rather modest. Whilst DES is unlikely to be used in pregnant women in the future, the findings of this study offer some reassurance regarding the potential of low-level exposure to environmental estrogens to affect human male reproductive development, given their extremely low potency compared with DES and the high exposures that resulted from therapeutic use of DES.

References

  • Full study (free access) : Effect of environmental and pharmaceutical exposures on fetal testis development and function: a systematic review of human experimental data, Human Reproduction Update, doi.org/10.1093/humupd/dmz004, 14 March 2019.
  • Featured image dmz004.pdf.
DES DIETHYLSTILBESTROL RESOURCES

Impact of environmental endocrine disrupting chemicals on the development of obesity

Developmental effects of diethylstilbestrol DES on obesity

2010 Review Abstracts

Environmental chemicals with hormone-like activity can disrupt programming of endocrine signaling pathways during development and result in adverse effects, some of which may not be apparent until much later in life. Recent reports link exposure to environmental endocrine disrupting chemicals during development with adverse health consequences, including obesity and diabetes. These particular diseases are quickly becoming significant public health problems and are fast reaching epidemic proportions worldwide. This review summarizes data from experimental animals and humans which support an association of endocrine disrupting chemicals, such as diethylstilbestrol, bisphenol A, phytoestrogens, phthalates, and organotins, with the development of obesity. Potential mechanisms are summarized and future research needs are discussed.

Also, the developing fetus and neonate have increased metabolic rates as compared to adults which in some cases may make them more vulnerable to chemical toxicity. It is now well established in the fields of nutrition and endocrine disruption that exposure to environmental chemicals during development can interfere with complex differentiating endocrine signaling pathways and cause adverse consequences later in life; the well known reproductive tract toxicity of diethylstilbestrol (DES) is one of the best examples of adverse consequences of endocrine disrupting chemicals. The concept of the “developmental origins of adult disease”, as the term implies, suggests that there is a time lag between exposure and manifestation of disease. In other words, the effects of exposure during development may not be readily apparent until much later in life.

DES, a potent synthetic estrogen, was widely prescribed to pregnant women from the 1940s through the 1970s in the mistaken belief that it could prevent threatened miscarriages. It was estimated that a range of 2 to 8 million pregnancies worldwide were exposed to DES. Today, it is well known that prenatal DES exposure resulted in a low but significant increase in neoplastic lesions and a high incidence of benign lesions in both the male and female offspring. The DES paradigm was a clear example that prenatal exposure could lead to adult-onset disease. To study the mechanisms involved in DES toxicity, we developed experimental mouse models of perinatal (prenatal or neonatal) DES exposure in which outbred mice were treated with DES on days 9-16 of gestation (the period of major organogenesis in the mouse) or days 1-5 of neonatal life (a period of cellular differentiation of the reproductive tract and a critical period of immune, behavioral, and adipocyte differentiation). These perinatal DES animal models have successfully duplicated, and in some cases predicted, many of the alterations (structural, functional, cellular, and molecular) observed in similarly DES-exposed humans. Further, these models have also shown multigenerational transmission of disease patterns implicating epigenetic mechanisms in the transmission of these effects.

Although our initial focus was on reproductive tract abnormalities and subfertility/infertility, we subsequently examined the relationship of perinatal DES treatment with the development of obesity later in life. We wanted to determine if DES was an “obesogen” as well as a reproductive toxicant and, if so, what were its molecular targets and the mechanisms through which it might act. For our obesity experiments, mice were treated with DES on days 1-5 of neonatal life using a low dose of 0.001 mg/day (1 µg/kg/day); this dose did not affect body weight during treatment but was associated with a significant increase in body weight in the adult animal by 4 to 6 months of age; male mice treated as neonates did not have an increase in body weight.

Unlike the low dose of DES (0.001 mg/day = 1 µg/kg/day), a higher dose (1000 µg/kg/day =1 mg/kg/day) caused a significant decrease in body weight during treatment but it was followed by a “catch-up” period around puberty and then finally resulted in an increase in body weight of the DES-treated mice compared to controls after ~2 months of age. This “catch-up” in weight between treated and control animals is reminiscent of the thrifty phenotype which is a well known phenomenon in the field of nutrition and was described in human infants who received poor nutrition during fetal life but later had “catch-up” growth that finally resulted in overweight and obesity later in life. Further studies indicated that the increase in body weight in DES-exposed mice was associated with an increase in the percent of body fat as determined by mouse densitometry.

Increased body weight in both low and high DES-treated mice compared to controls was observed throughout adulthood; however, by 18 months of age, statistical differences in body weight between DES-treated mice and controls were difficult to show because individual animal variability within groups increased as they aged due to the altered health status of the DES animals. We concluded that since various doses of DES resulted in obesity whether or not pups were underweight during treatment, multiple pathways might be involved in the programming for obesity related to environmental estrogens.
Since densitometry images of DES-treated mice suggested excessive abdominal fat, specific fat pads were weighed to see if particular fat pads were affected by DES treatment or whether there was a generalized change throughout the mouse, since it is well known that increased abdominal fat is associated with cardiovascular disease and diabetes in humans. Weights of inguinal, parametrial, gonadal, and retroperitoneal fat pads were all increased in DES treated mice as compared to controls at 6-8 months of age, suggesting a potential impact on cardiovascular disease following developmental exposure to DES. Brown fat weights were not significantly different in these animals.

Examination of DES-treated mice (1000 µg/kg/day =1 mg/kg/day) and controls at 2 months of age, prior to the treated mice becoming overweight and obese, showed elevated serum levels of leptin, adiponectin, IL-6, and triglycerides, suggesting that these endpoints may be important early markers of subsequent adult disease. Elevated levels of leptin are not surprising considering the increase number and size of the adipocytes in the DES-treated mice, but the increase in adiponectin was not expected since low levels usually correlate with obesity and diabetes in humans. This may indicate insensitivity to these hormones and/or a loss of the negative feedback mechanisms that regulate adipogenesis. Nevertheless, additional studies are needed to determine the mechanisms involved. At 6 months of age, insulin and all of the serum markers except triglycerides were found to be significantly elevated as compared to controls.

Glucose levels were also measured in DES (1000 µg/kg/day =1 mg/kg/day) and control mice at 2 months of age prior to the development of obesity and excessive weight gain. Interestingly, 25% of the DES-treated mice had significantly higher glucose levels than controls; these mice also showed a slower clearance rate of glucose from the blood since higher levels were seen throughout the experiment. Additional glucose measurements in older mice may help determine if a higher percentage of mice are affected with age and if higher and sustained levels of glucose can be demonstrated. To date, our data suggest that overweight and obesity observed in perinatal DES-treated mice will be associated with the development of diabetes, similar to the association of obesity with diabetes in humans. Further studies from our laboratory support a role for altered glucose metabolism as we have shown a high prevalence of islet cell hyperplasia in mice exposed to DES or other environmental estrogens including BPA and genistein.

Since the imbalance of activity levels and food intake are known contributors to obesity, ambulatory activity was measured in DES (1000 µg/kg/day =1 mg/kg/day) and control mice at 2 months of age before a difference in body weight could be detected. Overall, no statistical difference could be shown in activity between groups, although the DES group showed slightly less movement as compared to controls. This slight difference was not sufficient to explain the enhanced weight gain in DES mice as they aged.

Food consumption was also assessed; DES-treated mice consumed more than controls over the course of the experiment (~3 grams more), but the amounts were not statistically different between the groups. Taking into account both the marginal decrease in activity and the increase in food intake in DES-treated mice as compared to controls, it is unlikely that these two parameters can solely explain the development of obesity in DES-treated mice.

A recent study indicated a role for developmental genes in the origins of obesity and body fat distribution in mice and humans. We examined whether exposure to environmental chemicals which exerted hormonal activity would alter expression of genes involved in programming adipocytes during development. Several genes were found to be implicated in altered adipocyte differentiation and function (Hoxa5, Gpc4, and Tbx15) as well as fat cell distribution (Thbd, Nr2f1, and Sfrp2). We investigated changes in gene expression by microarray analysis in uterine samples from DES-treated mice (1000 µg/kg/day =1 mg/kg/day) compared to controls at 19 days of age. Genes involved in adipocyte differentiation were not different in the uterus following neonatal DES exposure. However, genes involved in fat distribution were altered; Thbd and Nr2f1 were significantly down-regulated and Sfrp2 was significantly up-regulated in DES-treated uteri compared to controls. These findings support the idea that environmental estrogens may play a role in regulating the expression of obesity-related genes in development. The identification of genes and molecular mechanisms that may be associated with EDCs and obesity is an exciting area of new research.

Although only neonatal exposure to DES has been discussed thus far in this review, exposure during prenatal life has also been shown to be associated with obesity later in life. Interestingly, high prenatal DES doses caused lower birth weight compared to controls, followed by a “catch-up period”, finally resulting in obesity; low prenatal DES doses had no effect on birth weight but it still resulted in obesity later in life. Thus, it appears that the effects of DES on adipocytes may depend on the time of exposure and the dose and that multiple mechanisms may be altered and result in the same obesity phenotype. Other investigators have also reported analogous findings with DES and other estrogenic chemicals.

References

  • Full study (free access) : Impact of environmental endocrine disrupting chemicals on the development of obesity, Hormones, PMID: 20688618, 2010 Jul-Sep.
  • Featured image PMC1931509/figure/F6.
DES DIETHYLSTILBESTROL RESOURCES

Perinatal exposure to DES linked to obesity increase in a sex-dependent manner

The endocrine disruptor diethylstilbestrol induces adipocyte differentiation and promotes obesity in mice

2012 Study Abstract

Epidemiology studies indicate that exposure to endocrine disruptors during developmental “window” contributes to adipogenesis and the development of obesity.

Implication of endocrine disruptor such as diethylstilbestrol (DES) on adipose tissue development has been poorly investigated.

Here we evaluated the effects of DES on adipocyte differentiation in vitro and in vivo, and explored potential mechanism involved in its action.

DES induced 3T3-L1 preadipocyte differentiation in a dose-dependent manner, and activated the expression of estrogen receptor (ER) and peroxisome proliferator-acivated receptor (PPAR) γ as well as its target genes required for adipogenesis in vitro. ER mediated the enhancement of DES-induced PPARγ activity. Moreover, DES perturbed key regulators of adipogenesis and lipogenic pathway in vivo.

In utero exposure to low dose of DES significantly increased body weight, liver weight and fat mass in female offspring at postnatal day (PND) 60. In addition, serum triglyceride and glucose levels were also significantly elevated.

These results suggest that perinatal exposure to DES may be expected to increase the incidence of obesity in a sex-dependent manner and can act as a potential chemical stressor for obesity and obesity-related disorders.

References

  • The endocrine disruptor diethylstilbestrol induces adipocyte differentiation and promotes obesity in mice, Toxicology and applied pharmacology, PMID: 22710028, 2012.
  • Featured image trbimg.
DES DIETHYLSTILBESTROL RESOURCES

Endocrine disruptors and obesity: obesogens

Developmental effects of diethylstilbestrol and other EDCs on obesity

Abstract

Incidence and prevalence of owerweight and obesity have greatly increased over the past three decades in almost all countries around the world.

This phenomenon is not easily explained by lifestyle changes in populations with very different initial habits. This has led to consider the influence of other factors, the so-called endocrine disruptors, and more specifically obesogens.

This study reviewed the available evidence about polluting chemical substances which may potentially be obesogens in humans: DES, genistein, bisphenol A, organotins (TBT, TPT), and phthalates. The first three groups of substances mainly act upon estrogen receptors, while organotins and phthalates activate PPARγ.

It was concluded that evidence exists of the obesogenic effect of these chemical substances in tissues and experimental animals, but few data are available in humans.

References

DES DIETHYLSTILBESTROL RESOURCES

Developmental effects of diethylstilbestrol and other EDCs on obesity

Developmental exposure to endocrine-disrupting chemicals programs for reproductive tract alterations and obesity later in life

Abstract

Many chemicals in the environment, especially those with estrogenic activity, are able to disrupt the programming of endocrine signaling pathways established during development; these chemicals are referred to as endocrine-disrupting chemicals. Altered programming can result in numerous adverse consequences in estrogen-target tissues, some of which may not be apparent until later in life. For example, a wide variety of structural, functional, and cellular effects have been identified in reproductive tract tissues. In addition to well-documented reproductive changes, obesity and diabetes have joined the list of adverse effects that have been associated with developmental exposure to environmental estrogens and other endocrine-disrupting chemicals.

Obesity is a significant public health problem reaching epidemic proportions worldwide. Experimental animal studies document an association of developmental exposure to environmental estrogens and obesity. For example, a murine model of perinatal exposure to diethylstilbestrol has proven useful in studying mechanisms involved in abnormal programming of differentiating estrogen-target tissues, including reproductive tract tissues and adipocytes. Other environmental estrogens, including the environmental contaminant bisphenol A, have also been linked to reproductive problems and obesity later in life. Epidemiology studies support similar findings in humans, as do studies of cells in culture.

Together, these findings suggest new targets for abnormal programming by estrogenic chemicals and provide evidence supporting the scientific concept termed the developmental origins of adult disease. Furthermore, the association of environmental estrogens with obesity and diabetes expands the focus on these diseases from intervention or treatment to include prevention or avoidance of chemical modifiers, especially during critical windows of development.

Developmental effects of diethylstilbestrol and other EDCs on obesity

Obesity and overweight have dramatically increased in prevalence in wealthy industrialized countries over the past 2 to 3 decades and also in poorer underdeveloped nations, where it often coexists with undernutrition. Obesity has now reached epidemic proportions in the United States, although a recent study found that its increase has stopped its upward spiral in the past few years; however, there is no indication of any decreases in prevalence. Common causes of obesity have usually been attributed to high-calorie, high-fat diets and a lack of exercise combined with a genetic predisposition for the disease. However, the current alarming rise in obesity cannot be solely explained by only these factors; an environmental component must be involved. It has been suggested that exposure to EDCs during critical stages of adipogenesis is contributing to the obesity epidemic. The term obesogens has been coined for environmental chemicals that stimulate fat accumulation, referring to the idea that they inappropriately regulate lipid metabolism and adipogenesis to promote obesity.

Experimental animal studies support the idea of involvement of EDCs in obesity; developmental exposure to numerous chemicals — including diethylstilbestrol, other estrogens, and other chemicals, such as tributyl tin — has been associated with obesity or overweight and adipogenesis. Recently, there has been much interest in the chemical bisphenol A (BPA) because of its high production volume and its potential for widespread environmental contamination. Numerous studies have now shown an association of BPA exposure with increased body weight and adiposity. The later study suggests that an increase in body weight is sex specific, but that timing and dose may contribute to the complexity of these findings because other investigators report effects in both males and females. Interestingly, a recent article describes similar increases, as previously reported, in the body weights of pups obtained from moms fed BPA in their diets during pregnancy; the doses were low and were considered “ecologically relevant” at 1 μg BPA/kg diet (1 ppb). However, unlike previous reports, the differences in body weight at weaning disappear as the mice age. This is probably due to the palatability of the diet, which was substituted at weaning because both control and BPA mice did not continue to gain weight on the new diets.

In vitro studies with BPA provide additional evidence of a role for this chemical in the development of obesity and further suggest specific targets; BPA causes 3T3-L1 cells (mouse fibroblast cells that can differentiate into adipocytes) to increase differentiation and, in combination with insulin, accelerates adipocyte formation. Other in vitro studies have shown that low doses of BPA, similar to diethylstilbestrol, impair calcium signaling in pancreatic α cells, disrupt β cell function, and cause insulin resistance (48, 49). Low environmentally relevant doses of BPA have also been reported to inhibit adiponectin and stimulate the release of inflammatory adipokines, such as interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α), from human adipose tissue, which suggests that BPA is involved in obesity and the related metabolic syndrome. Furthermore, other studies have linked BPA exposure to disruption of pancreatic β cell function and blood glucose homeostasis in mice, which suggests changes indicative of the metabolic syndrome.

Epidemiologic studies also support an association of BPA with obesity. BPA was detected at higher concentrations in both nonobese and obese women with polycystic ovarian syndrome than in nonobese healthy women, which suggests the possible involvement of BPA in polycystic ovarian syndrome and/or obesity.

References

  • Full study (free access) : Developmental exposure to endocrine-disrupting chemicals programs for reproductive tract alterations and obesity later in life, The American journal of clinical nutrition, PMC3364077, 2011 Dec.
  • Featured image Sandra Cohen-Rose and Colin Rose..
DES DIETHYLSTILBESTROL RESOURCES

Environmental Estrogens and Obesity

The Developmental Exposed DES Animal Model to Study Obesity, 2009

Abstract

Many chemicals in the environment, in particular those with estrogenic activity, can disrupt the programming of endocrine signaling pathways that are established during development and result in adverse consequences that may not be apparent until much later in life. Most recently, obesity and diabetes join the growing list of adverse consequences that have been associated with developmental exposure to environmental estrogens during critical stages of differentiation. These diseases are quickly becoming significant public health issues and are fast reaching epidemic proportions worldwide. In this review, we summarize the literature from experimental animal studies documenting an association of environmental estrogens and the development of obesity, and further describe an animal model of exposure to diethylstilbestrol (DES) that has proven useful in studying mechanisms involved in abnormal programming of various differentiating estrogen- target tissues. Other examples of environmental estrogens including the phytoestrogen genistein and the environmental contaminant Bisphenol A are also discussed. Together, these data suggest new targets (i.e., adipocyte differentiation and molecular mechanisms involved in weight homeostasis) for abnormal programming by estrogenic chemicals, and provide evidence that support the scientific hypothesis termed “the developmental origins of adult disease”. The proposal of an association of environmental estrogens with obesity and diabetes expands the focus on the diseases from intervention/treatment to include prevention/avoidance of chemical modifiers especially during critical windows of development.

The developmental exposed DES animal model to study obesity

DES, a potent synthetic estrogen, was widely prescribed to pregnant women from the 1940s through the 1970s with the mistaken belief that it could prevent threatened miscarriages. It was estimated that a range of 2 to 8 million pregnancies worldwide were exposed to DES. Today, it is well known that prenatal DES treatment resulted in a low but significant increase in neoplastic lesions, and a high incidence of benign lesions in both the male and female offspring exposed during fetal life. To study the mechanisms involved in DES toxicity, we developed experimental mouse models of perinatal (prenatal or neonatal) DES exposure over 30 years ago. Outbred CD-1 mice were treated with DES by subcutaneous injections on days 9–16 of gestation (the period of major organogenesis in the mouse) or days 1–5 of neonatal life (a period of cellular differentiation of the reproductive tract, and a critical period of immune, behavioral, and adipocyte differentiation). These perinatal DES animal models have successfully duplicated, and in some cases, predicted, many of the alterations (structural, function, cellular and molecular) observed in similarly DES- exposed humans.

Although our major focus was initially on reproductive tract abnormalities and subfertility/infertility, we also examined the relationship of perinatal DES treatment with the development of obesity later in life. We sought to determine if DES was an obesogen as well as a reproductive toxicant, and if so, what were its molecular targets and the mechanisms through which it might act. For our obesity experiments, mice were treated with DES on days 1–5 of neonatal life using a low dose of 0.001 mg/day (1 μg/kg/day); this dose did not affect body weight during treatment but was associated with a significant increase in body weight as adults. The featured image is a representative photomicrograph of control and neonatal DES treated female mice at 4–6 months of age; male mice treated as neonates did not demonstrate this increase in body weight. Unlike the lower dose of DES (0.001 mg/day = 1 μg/kg/day), a higher dose of DES (1000 μg/kg/day =1 mg/kg/day) caused a significant decrease in body weight during treatment which was followed by a “catch up” period around puberty and then finally resulted in an increase in body weight of the DES treated mice compared to controls after ~2 months of age. This is reminiscent of the thrifty phenotype described earlier for humans. Additional studies indicated that the increase in body weight in these DES-exposed mice was associated with an increase in the percent of body fat as determined by mouse densitometry.

Increased body weight in all DES treated mice, both low and high doses, was maintained throughout adulthood; however, by 18 months of age, statistical differences in body weight between DES treated and controls were difficult to show because individual animal variability within groups increased so much as they aged (data not included). Since various doses of DES resulted in obesity whether or not pups were underweight during treatment, most likely multiple pathways are involved in programming for obesity by environmental estrogens.

Densitometry images suggested DES treated mice had excessive abdominal fat which had been previously reported to be associated with cardiovascular disease and diabetes (Gillum 1987), therefore, we determined the weights of various fat pads to determine if specific fat pads were affected by DES treatment or whether it was a generalized change throughout the mouse. Fat pad weights were compared in DES treated mice (1000 μg/kg/day =1 mg/kg/day) and controls at 6–8 months of age; inguinal, parametrial, gonadal, and retroperitoneal fat pads were all increased in DES treated mice as compared to controls, but, brown fat weights were not significantly different at this age.

Although DES- treated mice were not statistically different in weight to controls at 2 months of age, DES (1000 μg/kg/day =1 mg/kg/day) mice exhibited elevated serum levels of leptin, adiponectin, IL-6, and triglycerides prior to becoming overweight and obese. This suggests that these endpoints may be important early markers of subsequent adult disease. The elevated levels of leptin are not surprising considering the increase number and size of the adipocytes in the DES treated mice but the increase in adiponectin was not expected since low levels usually correlate with diabetes. However, this may indicate insensitivity to these hormones and/or a loss of the negative feedback mechanisms that regulate adipogenesis. At 6 months of age, insulin and all of the serum markers except triglycerides were found to be significantly elevated as compared to controls.

Glucose levels were also measured in DES (1000 μg/kg/day =1 mg/kg/day) and control mice prior to the development of obesity at approximately 2 months of age (Newbold et al. 2007). Interestingly, 25% of the DES-treated mice had significantly higher glucose levels than controls; these mice also showed a slower clearance rate of glucose from the blood since higher levels were seen throughout the experiment. It is important to note that altered glucose levels were observed in these mice before they developed excessive weight. Additional glucose measurements in older mice may help determine if a higher percentage of mice are affected with age, and if higher and sustained levels of glucose can be demonstrated. To date, however, our data suggest that overweight and obesity observed in perinatal DES- treated mice will be associated with the development of diabetes, similar to the association of obesity with diabetes in humans. Earlier studies from our laboratory support a role for altered glucose metabolism since we have shown a high prevalence of islet cell hyperplasia in the pancreas of mice exposed to DES or other environmental estrogens including BPA and genistein treated mice.

Since, the balance of activity levels and food intake are known contributors to obesity, activity was measured in DES (1000 μg/kg/day =1 mg/kg/day) and control mice at 2 months of age before a difference in body weight could be detected. Individual mice were placed in an Opto-Max motor activity chamber (Columbus Instruments, Columbus, OH) and their ambulatory activity measured. Overall, there was no statistical difference in this parameter between the two groups although the DES group showed less movement as compared to controls as the experiment progressed. This difference, however, was not sufficient to explain the enhanced weight gain in DES mice as they age. Additional measures of activity including the running wheel measured during the dark photoperiod are being determined and are necessary before the role of activity in the development of obesity can be fully accessed.

Feed consumption was also measured in control and DES-treated mice (1000 μg/kg/day =1 mg/kg/day). DES-treated mice consumed more than controls over the course of the experiment (~3 grams more), but the amounts were not statistically different between the groups. Taken into account, both the marginal decrease in activity and the increase in food intake in DES treated mice as compared to controls, it is unlikely these two measurements can solely explain the development of obesity in DES treated mice.

A recent study describes a role for developmental genes in the origins of obesity and body fat distribution in mice and humans. Therefore, exposure to environmental chemicals with hormonal activity may be altering gene expression involved in programming adipocytes during development. Several genes have been implicated in altering adipocyte differentiation and function such as Hoxa5, Gpc4 and Tbx15 and fat cell distribution such as Thbd, Nr2f1 and Sfrp2. We investigated changes in gene expression by microarray analysis in uterine samples from DES treated mice (1000 μg/kg/day =1 mg/kg/day) compared to controls at 19 days of age. In these samples, genes involved in adipocyte differentiation were not altered in the uterus following neonatal DES exposure, however, genes involved in fat distribution were. Thbd and Nr2f1 were significantly down regulated and Sfrp2 was significantly up regulated in DES treated uteri compared to controls. These findings support the idea that environmental estrogens may play a role in regulating the expression of obesity-related genes in development.

Although the data summarized in this review describes only neonatal exposure to a high dose of DES, lower doses and exposure during prenatal life have also been shown to be associated with obesity later in life. Interestingly, high prenatal DES doses caused lower birth weight compared to controls, followed by a “catch-up period”, and finally resulted in obesity; low prenatal DES doses had no effect on birth weight but it still resulted in obesity later in life. Thus, it appears that the effects of DES on adipocytes may depend on the time of exposure and the dose, and that multiple mechanisms maybe altered resulting in the same obesity phenotype.

References

  • Full study (free access) : Environmental Estrogens and Obesity, Molecular and cellular endocrinology, PMC2682588, 2009 May 25.
  • Featured image PMC2682588/figure/F1.
DES DIETHYLSTILBESTROL RESOURCES

Effects of endocrine disruptors on obesity

The Developmental Exposed DES Animal Model to Study Obesity, 2008

Summary

Environmental chemicals with hormone-like activity can disrupt the programming of endocrine signalling pathways that are established during perinatal life and result in adverse consequences that may not be apparent until much later in life. Increasing evidence implicates developmental exposure to environmental hormone mimics with a growing list of adverse health consequences in both males and females. Most recently, obesity has been proposed to be yet another adverse health effect of exposure to endocrine disrupting chemicals (EDCs) during critical stages of development. Obesity is quickly becoming a significant human health crisis because it is reaching epidemic proportions worldwide, and is associated with chronic illnesses such as diabetes and cardiovascular disease. In this review, we summarize the literature reporting an association of EDCs and the development of obesity, and further describe an animal model of exposure to diethylstilbestrol that has proven useful in studying mechanisms involved in abnormal programming of various oestrogen target tissues during differentiation. Together, these data suggest new targets (i.e. adipocyte differentiation and mechanisms involved in weight homeostasis) of abnormal programming by EDCs, and provide evidence that support the scientific term ‘the developmental origins of adult disease’. The emerging idea of an association of EDCs and obesity expands the focus on obesity from intervention and treatment to include prevention and avoidance of these chemical modifiers.

The developmental exposed DES animal model to study obesity

The role of environmental chemicals in the development of obesity is an emerging area of research which is focusing on the identification of obesogens, their possible molecular targets and potential cellular mechanisms through which they might act. Thus, to determine if environmental chemicals with hormone‐like activity are playing a role in the development of obesity and, further, study potential mechanisms involved, we used an experimental mouse model of perinatal DES exposure which was developed and characterized in our laboratory to study altered developmental programming of the reproductive tract which is well known to result in disease and dysfunction.

To study the mechanisms involved in DES toxicity, we developed an animal model using outbred CD‐1 mice treated with DES by subcutaneous injections on days 9–16 of gestation (the period of major organogenesis in the mouse) or days 1–5 of neonatal life (a period of cellular differentiation of the reproductive tract and a critical period of immune, behavioral and adipocyte differentiation). The developmental DES animal model has been used to successfully duplicate, and in some cases, predict, many of the alterations (structural, function, cellular and molecular) observed in similarly DES‐exposed humans.

Although our major focus has been on reproductive tract abnormalities and subfertility/infertility, we also examined the effects of DES on body weight. Treatment of female mice with DES on days 1–5 of neonatal life using a low dose of 0.001 mg/day did not affect body weight during treatment but was associated with a significant increase in body weight as adults. Further, data indicated that the increase in body weight in these DES‐exposed mice was associated with an increase in the percent of body fat as determined by mouse densitometry. Featured image schematically shows the weight patterns of low dosed DES‐treated mice and controls.

Unlike the low dose of DES (0.001 mg/kg), the high neonatal DES dose of 1 mg/day caused a significant decrease in body weight of female mice during treatment on days 1–5 but this was followed by a ‘catch up’ period lasting until about 2 months of age and then finally resulting in a significant increase in body weight of DES‐treated mice as compared with controls. The high body weight in all DES‐treated mice, both low and high doses, was maintained throughout adulthood. These data suggest that numerous pathways are probably involved in programming for obesity because DES at different doses resulted in obesity whether or not pups were under weight during treatment.

As the densitometry images suggested DES‐treated mice had excessive abdominal fat which has been previously reported to be associated with cardiovascular disease and diabetes, weights of various fat pads were measured to determine if specific fat pads were affected by DES treatment or whether it was a generalized effect throughout the mouse. At 6–8 months of age, fat pad weights were compared in DES‐treated mice (1 mg/kg) and controls; inguinal, parametrial, gonadal and retroperitoneal fat pads were all increased in DES‐treated mice as compared with controls; however, brown fat weights were not significantly different between DES and controls.

A recent study describes a role for developmental genes in the origins of obesity and body fat distribution in mice and humans. Therefore, exposure to environmental chemicals with hormonal activity may be altering gene expression involved in programming adipocytes. Several genes have been implicated in altering adipocyte distribution and function such as Hoxa5, Gpc4 and Tbx15 and fat cell distribution such as Thbd, Nr2f1 and Sfrp2. We investigated changes in gene expression by microarray analysis in uterine samples from DES‐treated mice (1 mg/kg) compared with controls at 19 days of age. In these samples, genes involved in adipocyte distribution were not altered in the uterus following neonatal DES exposure, however, genes involved in fat distribution were. Thbd and Nr2f1 were significantly downregulated and Sfrp2 was significantly upregulated in DES‐treated uteri compared with controls. These findings support the idea that environmental oestrogens may play a role in regulating the expression of obesity‐related genes in development.

Although DES‐treated mice were similar in weight to controls at 2 months, high dose DES (1 mg/kg) mice exhibited elevated serum levels of leptin, adiponectin, IL‐6 and triglycerides before overweight and obesity developed suggesting these endpoints may be important early markers of subsequent adult disease. The elevated levels of leptin and adiponectin may indicate insensitivity to these hormones and/or a loss of the negative feedback mechanisms that regulate adipogenesis. Further, at 6 months of age, insulin and all of the serum markers except triglycerides were found to be significantly elevated as compared with controls.

As, the balance of activity levels and food intake are known contributors to obesity, activity was measured in DES (1 mg/day) and control mice at 2 months of age before a difference in body weight could be detected. Individual mice were placed in an Opto‐Max motor activity chamber (Columbus Instruments, Columbus, OH, USA) and their ambulatory activity measured. Overall, there was no statistical difference in this parameter between the two groups although the DES group showed less movement as compared with controls as the experiment progressed. This difference, however, was not sufficient to explain the enhanced weight gain in DES mice as they aged. Additional measures of activity will be necessary before decrease in activity can be ruled out as a contributing factor to the development of obesity in these mice.

Feed consumption was also measured over a 2‐week period for control and DES‐treated mice (1 mg/kg). Although DES‐treated mice ate more than controls over the course of the experiment (approximately 3 g more), the amounts were not statistically different from controls. Taken into account, both the marginal decrease in activity and the increase in food intake in DES‐treated mice as compared with controls, it is unlikely these two measurements can solely explain the development of obesity in DES‐treated mice.

Glucose levels were also measured in DES (1mg/kg) and control female mice at 2 months of age prior to the development of obesity (Newbold et al., 2007a,b). Twenty‐five percent of the DES‐treated mice had significantly higher glucose levels than controls; these mice also showed a slower clearance rate of glucose from the blood because higher levels were seen throughout the experiment. It is important to note that altered glucose levels were observed in these mice before they developed excessive weight. Perhaps additional glucose measurements in older mice may help determine if a higher percentage of mice are affected with age, and if higher and sustained levels of glucose can be demonstrated. To date, however, our data suggest that overweight and obesity observed in perinatal DES‐treated mice will be associated with the development of diabetes, similar to the association of obesity with diabetes in humans. Interestingly, earlier studies from our laboratory have shown a high prevalence of islet cell hyperplasia in the pancreas of DES‐treated mice supporting the idea that these mice have abnormal glucose metabolism.

Although female mice exposed to DES at low or high doses developed obesity as adults, these phenomena were not apparent in similarly exposed males. In fact, DES‐treated males were smaller than corresponding controls and the decrease weight was dose dependent. This sex‐specific effect is not surprising because organizational effects of sex steroids during critical developmental periods have been well known for years to cause changes in the long‐term anatomy and function of the hypothalamic nuclei which regulates reproduction as well as body weight. Certainly, additional studies are necessary to investigate the effects of EDCs on male weight homeostasis and adipocytes because it is not clear whether this is a compound‐specific effect, an oestrogenic effect or typical of EDCs with other hormonal activities. Again, our findings point out the complexities of the mechanisms associated with the development of obesity.

References

DES DIETHYLSTILBESTROL RESOURCES

Perinatal exposure to environmental estrogens and the development of obesity

Brief exposure early in life to DES increases body weight as the mice age

“We can no longer simply assume that overweight and obesity are just personal choices based on the foods we eat, but that complex events including exposure to environmental chemicals during development may be contributing to (the) obesity (epidemic).”

Abstract

Dietary substances and xenobiotic compounds with hormone-like activity can disrupt the programming of endocrine signaling pathways that are established during perinatal differentiation. The consequences of this disruption may not be apparent until later in life but increasing evidence implicates developmental exposure to environmental hormone-mimics with a growing list of adverse health effects including reproductive problems and increased cancer risks.

Obesity has recently been proposed to be yet another adverse health consequence of exposure to endocrine disrupting substances during development. There is a renewed focus on identifying contributions of environmental factors to the development of obesity since it is reaching worldwide epidemic proportions, and this disease has the potential to overwhelm healthcare systems with associated illnesses such as diabetes and cardiovascular disease.

Here, we review the literature that proposes an association of perinatal exposure to endocrine disrupting chemicals, in particular those with estrogenic activity, with the development of obesity later in life. We further describe an animal model of developmental exposure to diethylstilbestrol (DES) to study mechanisms involved in programming for obesity.

Our experimental data support the idea that adipocytes and the mechanisms involved in weight homeostasis are novel targets of abnormal programming of environmental estrogens, some of which are found in our foods as naturally occurring substances or inadvertently as contaminants.

References

  • Perinatal exposure to environmental estrogens and the development of obesity, Molecular nutrition & food research, PMID: 17604389, 2007 Jul.
  • Image credit commons.wikimedia.
DES DIETHYLSTILBESTROL RESOURCES