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 (Newbold 1995
). 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) (McLachlan et al. 1980
) or days 1–5 of neonatal life (Newbold 2004
; Newbold et al. 1990
) (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. 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 (Newbold et al. 2008
). 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. shows the weight patterns of DES treated (1000 μg/kg/day =1 mg/kg/day) mice and their corresponding controls. 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 (Lunar PIXIMUS, GE Healthcare, Waukesha, Wi) ( and ).
Representative photograph of Control and DES-treated Mice
Body Weight of Mice Following DES Exposure
Increased body fat in mice treated neonatally with DES as determined by PIXImus™ 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 ()(Newbold et al. 2005
Fat Pad Weights of Mice Treated Neonatally with DES
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 () (Newbold et al. 2007
Serum Profiles of Mice Treated Neonatally with DES
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 (Newbold et al. 2007
). 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 (unpublished).
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 (Newbold et al. 2007
). 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 (Newbold et al. 2007
). 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 (Gesta et al. 2006
). 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
and fat cell distribution such as Thbd
. 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
were significantly down regulated and Sfrp2
was significantly up regulated in DES treated uteri compared to controls (data taken from study published by (Newbold et al. 2007
). 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 (Newbold et al. 2005
). 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.