Frederick S. vom Saal

Low-dose bioactivity of xenoestrogens in animals: fetal exposure to low doses of methoxychlor and other xenoestrogens increases adult prostate size in mice.

The hormonal activity of natural estrogens is influenced by the degree to which they bind to serum proteins. In the pregnant female and in the fetus, greater than 99% of estradiol may be bound by serum binding proteins. Therefore, even though total serum levels of estradiol appear very high in fetuses, we have found that in rodent fetuses, there is a very low free concentration of estradiol (0.2 pg/ml). Naturally occurring variation in fetal serum estradiol predicts differences in numerous postnatal traits, including prostate size. In addition, when this low level of free estradiol was experimentally increased from 0.2 to 0.3 pg/ml during the last third of fetal life, treated male mice showed an increase in adult prostate weight. Fetal exposure to low doses of xenobiotic estrogens by feeding to pregnant females, including the compounds methoxychlor (20 and 2000 μg/kg body weight), DES (0.02 to 2 μg/kg body weight) and bisphenol A (2 and 20 μg/kg body weight), also led to increased prostate weight in adulthood. In contrast, fetal doses of natural estradiol and DES above the physiological range of estrogenic activity, and within a toxicological dose range, led to the opposite outcome, a reduction in subsequent adult prostate weight. This indicates that it may be impossible to assess endocrine-disrupting activities in response to low doses within a physiological range of activity by using high, toxic doses of xenoestrogens in testing procedures. We have developed approaches in vitro to predict the potential estrogenic bioactivity of compounds in the physiologically relevant range in animals and humans. We address the following factors in predicting the final observed endocrine-disrupting effect in the animal: (1) the intrinsic estrogenic activity of a given molecule, (2) the effective free concentration determined by how the molecule is carried in serum, (3) partitioning between aqueous and lipid compartments in body and cell lipids, and (4) absorption and metabolism relative to the route of exposure. The studies and strategies we describe are important in developing criteria for a tiered testing system for the detection of estrogenic chemicals as well as endocrine-disrupting chemicals with different modes of action.

A Clash of Old and New Scientific Concepts in Toxicity, with Important Implications for Public Health

Background A core assumption of current toxicologic procedures used to establish health standards for chemical exposures is that testing the safety of chemicals at high doses can be used to predict the effects of low-dose exposures, such as those common in the general population. This assumption is based on the precept that “the dose makes the poison”: higher doses will cause greater effects. Objectives We challenge the validity of assuming that high-dose testing can be used to predict low-dose effects for contaminants that behave like hormones. We review data from endocrinology and toxicology that falsify this assumption and summarize current mechanistic understanding of how low doses can lead to effects unpredictable from high-dose experiments. Discussion Falsification of this assumption raises profound issues for regulatory toxicology. Many exposure standards are based on this assumption. Rejecting the assumption will require that these standards be reevaluated and that procedures employed to set health standards be changed. The consequences of these changes may be significant for public health because of the range of health conditions now plausibly linked to exposure to endocrine-disrupting contaminants. Conclusions We recommend that procedures to establish acceptable exposure levels for endocrine-disrupting compounds incorporate the inability for high-dose tests to predict low-dose results. Setting acceptable levels of exposure must include testing for health consequences at prevalent levels of human exposure, not extrapolations from the effects observed in high-dose experiments. Scientists trained in endocrinology must be engaged systematically in standard setting for endocrine-disrupting compounds.

Regulatory decisions on endocrine disrupting chemicals should be based on the principles of endocrinology

For years, scientists from various disciplines have studied the effects of endocrine disrupting chemicals (EDCs) on the health and wellbeing of humans and wildlife. Some studies have specifically focused on the effects of low doses, i.e. those in the range that are thought to be safe for humans and/or animals. Others have focused on the existence of non-monotonic dose-response curves. These concepts challenge the way that chemical risk assessment is performed for EDCs. Continued discussions have clarified exactly what controversies and challenges remain. We address several of these issues, including why the study and regulation of EDCs should incorporate endocrine principles; what level of consensus there is for low dose effects; challenges to our understanding of non-monotonicity; and whether EDCs have been demonstrated to produce adverse effects. This discussion should result in a better understanding of these issues, and allow for additional dialog on their impact on risk assessment.

An Extensive New Literature Concerning Low-Dose Effects of Bisphenol A Shows the Need for a New Risk Assessment

Bisphenol A (BPA) is the monomer used to manufacture polycarbonate plastic, the resin lining of cans, and other products, with global capacity in excess of 6.4 billion lb/year. Because the ester bonds in these BPA-based polymers are subject to hydrolysis, leaching of BPA has led to widespread human exposure. A recent report prepared by the Harvard Center for Risk Analysis and funded by the American Plastics Council concluded that evidence for low-dose effects of BPA is weak on the basis of a review of only 19 studies; the report was issued after a delay of 2.5 years. A current comprehensive review of the literature reveals that the opposite is true. As of December 2004, there were 115 published in vivo studies concerning low-dose effects of BPA, and 94 of these report significant effects. In 31 publications with vertebrate and invertebrate animals, significant effects occurred below the predicted “safe” or reference dose of 50 μg/kg/day BPA. An estrogenic mode of action of BPA is confirmed by in vitro experiments, which describe disruption of cell function at 10−12 M or 0.23 ppt. Nonetheless, chemical manufacturers continue to discount these published findings because no industry-funded studies have reported significant effects of low doses of BPA, although > 90% of government-funded studies have reported significant effects. Some industry-funded studies have ignored the results of positive controls, and many studies reporting no significant effects used a strain of rat that is inappropriate for the study of estrogenic responses. We propose that a new risk assessment for BPA is needed based on a) the extensive new literature reporting adverse effects in animals at doses below the current reference dose; b) the high rate of leaching of BPA from food and beverage containers, leading to widespread human exposure; c) reports that the median BPA level in human blood and tissues, including in human fetal blood, is higher than the level that causes adverse effects in mice; and d) recent epidemiologic evidence that BPA is related to disease in women.

A Physiologically Based Approach To the Study of Bisphenol a and Other Estrogenic Chemicals On the Size of Reproductive Organs, Daily Sperm Production, and Behavior

Two chemicals previously shown to have estrogenic activity, bisphenol A and octylphenol, were examined for their effects on accessory reproductive organs and daily sperm production in male offspring of mice fed these chemicals during pregnancy. These chemicals are used in the manufacture of plastics and other products, and have been detected in food and water consumed by animals and people. From gestation day 11-17 female mice were fed an average concentration (dissolved in oil) of bisphenol A or octylphenol of 2 ng/g body weight (2 ppb) and 20 ng/g (20 ppb). The 2 ppb dose of bisphenol A is lower than the amount reported to be swallowed during the first hour after application of a plastic dental sealant (up to 931 μg; 13.3 ppb in a 70 kg adult). We found that the 2 ng/g dose of bisphenol A permanently increased the size of the preputial glands, but reduced the size of the epididymides; these organs develop from different embryonic tissues. At 20 ng/g, bisphenol A significantly decreased efficiency of sperm production (daily sperm production per g testis) by 20% relative to control males. The only significant effect of octylphenol was a reduction in daily sperm production and efficiency of sperm production at the 2 ng/g dose. A new approach to studying physiologically relevant doses of environmental endocrine disruptors is discussed, particularly with regard to the development of the reproductive organs, the brain, and behavior.

Our plastic age

Within the last few decades, plastics have revolutionized our daily lives. Globally we use in excess of 260 million tonnes of plastic per annum, accounting for approximately 8 per cent of world oil production. In this Theme Issue of Philosophical Transactions of the Royal Society, we describe current and future trends in usage, together with the many benefits that plastics bring to society. At the same time, we examine the environmental consequences resulting from the accumulation of waste plastic, the effects of plastic debris on wildlife and concerns for human health that arise from the production, usage and disposal of plastics. Finally, we consider some possible solutions to these problems together with the research and policy priorities necessary for their implementation.

Components of plastic: experimental studies in animals and relevance for human health

Components used in plastics, such as phthalates, bisphenol A (BPA), polybrominated diphenyl ethers (PBDE) and tetrabromobisphenol A (TBBPA), are detected in humans. In addition to their utility in plastics, an inadvertent characteristic of these chemicals is the ability to alter the endocrine system. Phthalates function as anti-androgens while the main action attributed to BPA is oestrogen-like activity. PBDE and TBBPA have been shown to disrupt thyroid hormone homeostasis while PBDEs also exhibit anti-androgen action. Experimental investigations in animals indicate a wide variety of effects associated with exposure to these compounds, causing concern regarding potential risk to human health. For example, the spectrum of effects following perinatal exposure of male rats to phthalates has remarkable similarities to the testicular dysgenesis syndrome in humans. Concentrations of BPA in the foetal mouse within the range of unconjugated BPA levels observed in human foetal blood have produced effects in animal experiments. Finally, thyroid hormones are essential for normal neurological development and reproductive function. Human body burdens of these chemicals are detected with high prevalence, and concentrations in young children, a group particularly sensitive to exogenous insults, are typically higher, indicating the need to decrease exposure to these compounds.

THE ESTROGENIC ENDOCRINE DISRUPTING CHEMICAL BISPHENOL A (BPA) AND OBESITY

There is increasing experimental and epidemiological evidence that fetal programming of genetic systems is a contributing factor in the recent increase in adult obesity and other components of metabolic syndrome. In particular, there is evidence that epigenetic changes associated with the use of manmade chemicals may interact with other factors that influence fetal and postnatal growth in contributing to the current obesity epidemic. The focus of this review is on the developmental effects of estrogenic endocrine disrupting chemicals (EDCs), and more specifically on effects of exposure to the estrogenic EDC bisphenol A (BPA), on adipocytes and their function, and the ultimate impact on adult obesity; BPA exposure also results in impaired reproductive capacity. We discuss the interaction of EDCs with other factors that impact growth during fetal and neonatal life, such as placental blood flow and nutrient transport to fetuses, and how these influence fetal growth and abnormalities in homeostatic control systems required to maintain normal body weight throughout life.

Why public health agencies cannot depend on good laboratory practices as a criterion for selecting data: The case of Bisphenol A

Background In their safety evaluations of bisphenol A (BPA), the U.S. Food and Drug Administration (FDA) and a counterpart in Europe, the European Food Safety Authority (EFSA), have given special prominence to two industry-funded studies that adhered to standards defined by Good Laboratory Practices (GLP). These same agencies have given much less weight in risk assessments to a large number of independently replicated non-GLP studies conducted with government funding by the leading experts in various fields of science from around the world. Objectives We reviewed differences between industry-funded GLP studies of BPA conducted by commercial laboratories for regulatory purposes and non-GLP studies conducted in academic and government laboratories to identify hazards and molecular mechanisms mediating adverse effects. We examined the methods and results in the GLP studies that were pivotal in the draft decision of the U.S. FDA declaring BPA safe in relation to findings from studies that were competitive for U.S. National Institutes of Health (NIH) funding, peer-reviewed for publication in leading journals, subject to independent replication, but rejected by the U.S. FDA for regulatory purposes. Discussion Although the U.S. FDA and EFSA have deemed two industry-funded GLP studies of BPA to be superior to hundreds of studies funded by the U.S. NIH and NIH counterparts in other countries, the GLP studies on which the agencies based their decisions have serious conceptual and methodologic flaws. In addition, the U.S. FDA and EFSA have mistakenly assumed that GLP yields valid and reliable scientific findings (i.e., “good science”). Their rationale for favoring GLP studies over hundreds of publically funded studies ignores the central factor in determining the reliability and validity of scientific findings, namely, independent replication, and use of the most appropriate and sensitive state-of-the-art assays, neither of which is an expectation of industry-funded GLP research. Conclusions Public health decisions should be based on studies using appropriate protocols with appropriate controls and the most sensitive assays, not GLP. Relevant NIH-funded research using state-of-the-art techniques should play a prominent role in safety evaluations of chemicals.

Similarity of bisphenol A pharmacokinetics in rhesus monkeys and mice: Relevance for human exposure

Objective Daily adult human exposure to bisphenol A (BPA) has been estimated at < 1 μg/kg, with virtually complete first-pass conjugation in the liver in primates but not in mice. We measured unconjugated and conjugated BPA levels in serum from adult female rhesus monkeys and adult female mice after oral administration of BPA and compared findings in mice and monkeys with prior published data in women. Methods Eleven adult female rhesus macaques were fed 400 μg/kg deuterated BPA (dBPA) daily for 7 days. Levels of serum dBPA were analyzed by isotope-dilution liquid chromatography–mass spectrometry (0.2 ng/mL limit of quantitation) over 24 hr on day 1 and on day 7. The same dose of BPA was fed to adult female CD-1 mice; other female mice were administered 3H-BPA at doses ranging from 2 to 100,000 μg/kg. Results In monkeys, the maximum unconjugated serum dBPA concentration of 4 ng/mL was reached 1 hr after feeding and declined to low levels by 24 hr, with no significant bioaccumulation after seven daily doses. Mice and monkeys cleared unconjugated serum BPA at virtually identical rates. We observed a linear (proportional) relationship between administered dose and serum BPA in mice. Conclusions BPA pharmacokinetics in women, female monkeys, and mice is very similar. By comparison with approximately 2 ng/mL unconjugated serum BPA reported in multiple human studies, the average 24-hr unconjugated serum BPA concentration of 0.5 ng/mL in both monkeys and mice after a 400 μg/kg oral dose suggests that total daily human exposure is via multiple routes and is much higher than previously assumed.