Durando, M, L Kass, J Piva, C Sonnenschein, AM Soto, EH Luque, and M Muñoz-de-Toro. 2006. Prenatal Bisphenol A Exposure Induces Preneoplastic Lesions in the Mammary Gland in Wistar Rats. Environmental Health Perspectives, in press
What did they do?
What did they find?
What does it mean?
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Prenatal exposure to bisphenol A (BPA) causes long-lasting changes in female rat breast tissue that make the tissue more sensitive to estrogen at puberty and more susceptible to cancer-causing chemicals as adults.
The findings add to existing evidence showing that long-term mammary gland changes can be caused by BPA exposure in the womb. These new results add a new twist: not only is mammary gland development altered in ways that increase risk of tumors, in utero exposure to low doses of BPA increases breast cancer risk by enhancing the vulnerability of the system to a known carcinogen.
The study strengthens support for a link between increasing rates of breast cancer in the last decades and increasing exposure to chemicals like BPA.
Prior studies have found prenatal exposure to BPA, a common ingredient in many everyday products, causes biochemical, cell, and tissue changes that lead to long-lasting effects in the breast and prostate. The chemical has been measured in human saliva, blood, urine, pregnant women and fetal blood, and the placenta at birth. According to data from the CDC, it can be detected in 95% of Americans.
The doses of BPA used by Durando et al. were chosen to be environmentally relevant: 25 µg/kg, or 25 parts per billion per day.
What did they do?
In one set of experiments, Durando et al. examined the effect of BPA exposure in the womb on how quickly animals reached puberty and on breast tissue development.
In a second set of experiments, they tested how BPA exposure in the womb interacts with adult exposure to a known carcinogen.
To deliver the BPA, Durando et al. implanted miniature pumps in the pregnant females on day 8 of pregnancy (D8), when internal organs of the fetus begin to develop. The pumps continuously released 0.25 µl/hour of a solution of that delivered 25 µg BPA/kg body weight per day into the experimental animals for 14 days (D8-D23). Controls received the implant but were treated only with the vehicle dimethylsulfoxide (DMSO).
Litters were delivered at D23 and weaned 21 days later.
In the first experiment, daughters were sacrificed at prepuberty (30 days old), post puberty (50 days old), and adulthood (110 and 180 days old) to determine if exposure to BPA in the womb affected development of the daughters' mammary gland tissue.
A second experiment examined whether prenatal exposure to BPA made the mammary tissue more sensitive to adult exposure to a carcinogen, N-nitroso-N-methylurea (NMU). When young adult female Wistar rats are injected once with 50 mg/kg NMU they are highly likely to develop malignant breast tumors. To set up this second experiment, Durando et al. first determined that 25 mg/kg NMU did not cause tumors by itself.
Rats received either a dose of 25 mg/kg of body weight (an amount which they had shown previously by itself does not cause cancer) or a dose known to cause cancer, 50 mg/kg. This latter dose served as the positive control.
Researchers examined the animals biweekly for tumors after they reached adulthood. Tissues of females sacrificed at 110 and 180 days old were also analyzed for tumors that were not detected by the biweekly external palpation.
Mammary gland tissue dissected from the female rats was analyzed for a number of tumor-associated breast cell anomalies. Samples of both stroma and parenchyma cell structures that make up the mammary gland were examined for abnormal cell death, cell proliferation (hyperplasia), stromal cell nuclei density, number of mast cells, and epithelial cells growing in stroma tissue.
What did they find?
None of the control animals (no BPA, no NMU) developed tumors. No animals treated with BPA alone developed tumors (although they did show changes in mammary gland tissues consistent with an increased risk of cancer). None of the animals treated with low levels of NMU and no BPA developed tumors.
The only animals to develop tumors were (1) those treated with high levels of NMU and (2) those treated with BPA in the womb and low levels of NMU at PND 50 (they did not test BPA and high NMU).
1. BPA treatment vs controls, without NMU.
They found several differences in rats exposed to BPA before birth compared to the control animals. Exposed females experienced early puberty (PND 34 vs PND 39 in controls) and even without NMU treatment had altered mammary tissue growth and development compared to controls. These tissue changes were only observed after puberty. The changes were consistent with an increased cancer risk.
For example, increased growth rate was observed in both parenchyma and stroma of the mammary gland in 50-day-old experimental rats. Slower programmed cell death (apoptosis) and a slight increase in cell proliferation attributed to the increased rates. Gland development was also affected by prenatal exposure.
Significant differences in the mammary glands of BPA-treated adult animals (PND 110 and PND 180) included a higher percentage of hyperplastic ducts (a precancerous condition), an increase in stromal nuclei density, and an increase density of mast cells surrounding the altered ducts. Mast cells are a multifunction immune cells associated with tumor initiation and growth; an increased density of mast cells around the ducts is thought to promote cancer.
Furthermore, stroma cells grew into surrounding epithelial layers and replaced normal fat cells in the mammary glands adipose tissue.
2. Effects of NMU, without BPA.
Animals treated with 25 mg/kg of NMU at PND 50 subsequently did not develop tumors. They did develop hyperplastic lesions.
Treatment with 50 mg/kg NMU at PND 50 did increase the number of precancerous ducts in older animals (PND 180) but not in young adults (PND 110).
3. BPA and NMU.
Hyperplastic ducts were twice as frequent (35% ±3.7) in animals exposed to BPA in the womb and subsequently (PND 50) to 25 mg/kg NMU, compared to those exposed only to NMU (15.7 ±1.2). The percentage of hyperplastic ducts in BPA+high NMU was also greater than the percentage in animals receiving only high NMU.
Two of seven females exposed to BPA in the womb and 25 mg/kg NMU at PND 50 had malignant cancers at PND 180. None of the 10 treated only with 25 mg/kg NMU developed tumors. Seven of 10 receiving high NMU only developed malignant cancers.
What does it mean?
These results support growing evidence that pre-birth exposure to estrogen-like compounds such as BPA increases the risk of breast cancer later in life.
Two findings are particularly relevant to this hypothesis: (1) the changes in tissue growth and development observed following BPA exposure; and (2) the impact of BPA exposure on subsequent sensitivity to a carcinogen.
(1) In these experiments, early exposure to environmentally-relevant levels of BPA increased pre-cancerous conditions in developing mammary cells and tissues. The tissue changes were evident by the time the animals reached puberty, when estrogen activity increases naturally.
The results on increased cell proliferation and decreased apoptosis indicate that BPA exposure during early development causes mammary glands of exposed rats to be more sensitive to estrogen, leading to unregulated tissue growth characteristic of cancer starting at puberty and persisting into adulthood. Accoring to Durondo et al. "the increased incident of hyperplastic ducts and increased stromal nuclear density observed in adult animals may be a consequence of the cellular turnover deregulation which occurred earlier in life, i.e. around puberty."
(2) In utero BPA increased sensitivity to the known carcinogen NMU, increasing the frequency of hyperplastic lesions and resulting in breast cancer at a level of NMU exposure that normally would not have caused malignant tumors. This 'two-hit' mechanism of carcinogenesis has been shown in multiple animal experiments, although not before with BPA.
The findings provide more supportive evidence for the hypothesis that increasing rates of human breast cancer in the past decades are related to increasing exposures to environmental estrogens like BPA.
They also severely challenge epidemiological studies that fail to incorporate developmental exposures. Most epidemiological studies of breast cancer risk in people have looked only at exposures measured after cancer diagnosis, for example, the well-known study of breast cancer on Long Island. Because these studies ignore biological mechanisms shown repeatedly in animal experiments, they can't be trusted to identify the causes of breast cancer in people.