American Scientist

The industrialized world produces an immense amount of plastic, more than 45 billion kilograms annually in the United States alone. But what is it made of, and is it all safe?

Some reusable water bottles sold in Wal-Mart and other retail stores in the United States now display stickers proudly marketing themselves as “BPA-free.” The labeling results from consumer concern over scientific evidence that bisphenol A (BPA), a common ingredient in many hard plastics, may be harmful to the human reproductive system because it interferes with hormones. The plastics industry and the U.S. Food and Drug Administration (FDA) say BPA is not dangerous at the levels people are currently exposed to. In contrast, in September of 2008, the U.S. National Toxicology Program (NTP) concluded that there is “some concern” for adverse effects on the “brain, behavior and prostate gland in fetuses, infants, and children.” This concern prompted members of Congress to pressure the FDA to take another look, a process that is now underway.

Inconsistent messages about BPA safety have generated considerable public rancor, highlighting how human risk assessment of BPA (and compounds like it) is both uncoordinated and controversial. Consensus regarding BPA’s safety has evaded U.S. health agencies for multiple reasons. Most pressing is the lack of clear guidelines for how much or what type of scientific evidence is needed to judge risks from hormone-disrupting compounds such as BPA. It would be unethical to directly assess those risks in people through controlled, double-blinded exposure experiments. At the same time, there are uncertainties about when exposure data from animal studies are relevant to human health. In a global environment where BPA production and exposure have grown rapidly, there is a pressing need to overcome these challenges. That is especially true because BPA is only one of thousands of chemicals thought to possibly have unintended effects on reproductive health.

In the U.S., BPA ranks in the top 2 percent of high-production-volume chemicals. BPA is a monomer that makes polycarbonate plastics harder and more resilient. Polycarbonate plastics are typically clear and often designated by a “7” within their triangular recycle symbols. BPA is also found in the epoxy resins that line the inside of metallic cans (such as soup and soda cans) and water storage tanks. BPA is also in thousands of other products, including compact discs, eyeglasses, thermal paper, polycarbonate water pipes, medical devices and dental sealants. Consumption is thought to be the most common route of human exposure because BPA readily leaches from food containers into their contents, especially when its heated, including in a microwave. Centers for Disease Control and Prevention (CDC) scientists have estimated that more than 92 percent of Americans have BPA in their bodies, and the highest levels are typically found in children.

BPA is an endocrine disruptor, which the Environmental Protection Agency (EPA) defines as “an exogenous chemical substance or mixture that alters the structure or function(s) of the endocrine system and causes adverse effects” in individuals, their offspring or populations. Hormones have many modes of action, including the ability to initiate or suppress gene transcription. Endocrine disruptors interfere with those genetic-level processes. The timing of exposure is critical because vulnerability changes over a lifespan. In an adult, where sex-specific physiology and behaviors have matured and function properly, interference with hormone action is likely reversible once exposure ends. This is not thought to be the case in the developing fetus or young child. Therefore, fetuses and infants are the groups most at risk for adverse effects from an endocrine disruptor such as BPA.

Over the course of development, hormones actually help shape the vertebrate brain and reproductive organs that allow sex-specific physiology and behaviors to emerge. Much of this happens in discrete developmental windows that span gestation through the postnatal period and, to some degree, into adolescence. Disruption of this developmental progression can permanently alter the organization of sexually dimorphic neuroendocrine circuits in the brain, the cellular composition of reproductive organs, the timing of puberty, the development of sex-typical behaviors and, ultimately, the capacity to reproduce. BPA exposure, even at levels humans are likely exposed to, appears to produce every one of these effects in laboratory rats and mice. But does it produce similar reproductive disorders in humans?

The reproductive effects found in laboratory rodents mirror some disturbing human health trends in industrialized nations. For example, an analysis of more than 100 research studies concluded that sperm counts in the United States and Europe appear to have declined by roughly half over the past 50 years. Researchers in Denmark now estimate that more than 10 percent of men in that country have sperm counts in the infertile range and up to 30 percent in the subfertile range. Rates of testicular cancer appear to be increasing. There are indications that female fecundity is declining, even among young women, although the rate and degree have been difficult to quantify. Median ages at menarche, first breast development and sexual precocity are dropping, especially among minority populations in the U.S. Similar trends have been noted in Europe and among children adopted from developing countries by parents in industrialized settings. The cause is likely complex and multifaceted, but the rapidity of the changes suggests an environmental component. Whether BPA might be involved hinges on whether effects observed in rodents reasonably predict what could be happening in humans.

The BPA Puzzle

BPA has more than one mode of action, but it is primarily recognized to be an estrogen mimic. Effects from endocrine disruption by an estrogen mimic in the developing fetus are likely subtle and not readily apparent at birth. The best example of this in humans is the tragic case of diethylstilbestrol (DES), a synthetic estrogen prescribed to upwards of 10 million women between 1938 and 1971. DES incorrectly was thought to reduce the risk of miscarriage. Instead, many children whose mothers took DES during pregnancy, both male and female, developed reproductive health problems as adults. Decades of DES use passed before indications of adverse effects were detected. A pair of physicians noted that DES daughters were more likely than unexposed women to develop an extremely rare form of vaginal clear cell carcinoma. Subsequently, malformations of the uterus, increased risk of testicular cancer, infertility and other reproductive health problems were also ascribed to fetal DES exposure. With DES, the severity of health effects corresponds to the timing and level of in utero exposure, illustrating the importance of tracking critical windows of exposure when attempting to predict potential consequences from human exposure to endocrine disruptors.

Important differences exist between DES and BPA. Most significantly, BPA is not as potent. The binding affinity of BPA for the two primary forms of estrogen receptor (ERα and ERβ) is approximately 10,000-fold weaker than that of estradiol, a natural estrogenic hormone, or of DES. To put this in context, the binding affinity of DDT and BPA are in a similar range. Also, human exposure to BPA is estimated to be relatively low—lower than the DES doses pregnant women received. Published estimates of human exposure to BPA are somewhat variable but generally fall in the range of 1 to 15 micrograms per kilogram of body weight per day or less, with infants and children on the upper end of that range and adults on the lower. Blood and urinary levels increase following the consumption of beverages from BPA-containing polycarbonate bottles, indicating that exposure rises and falls as people use products containing BPA. The EPA has set a “reference dose” of 50 micrograms per kilogram of body weight. This dose is considered safe for humans, even with daily exposure over a lifetime. It is based on the Lowest Observed Adverse Effect Level (LOAEL) of 50 milligrams per kilogram of body weight per day, the lowest dose at which any adverse effects should be observed in laboratory animals. BPA’s low estrogenic potency combined with the low level of human exposure have traditionally been interpreted to indicate little to no risk of human health effects from BPA exposure.

Yet there is growing evidence from multiple laboratories, including ours, that BPA can alter rodent reproductive physiology and behavior at doses equivalent or below the current LOAEL and, even more disturbingly, below the reference dose for humans. One of the most frequently observed consequences of low-dose exposure during development is the premature loss of a regular estrous cycle, an effect that produces an abbreviated period of fertility. We found that female rats exposed to the reference dose of 50 micrograms of BPA per kilogram for only the first four days of life developed an irregular estrous cycle as young as two and a half months of age. Rats normally remain fertile for upwards of a year. Similar effects have been reported in both rats and mice at equivalent or even lower doses.

Another well-documented effect in female rodents, observed by us and others, is disruption of pubertal timing. The effect is dose dependent: Doses near the 50-micrograms-per-kilogram reference dose advance pubertal onset, whereas higher doses delay it. Disruption of puberty is of particular interest for two primary reasons. First, it appears to be induced by lower, rather than higher, BPA doses, the mechanism for which is not well understood. Second, there is increasing evidence that puberty in girls is occurring earlier, especially in industrialized countries. Girls with advanced puberty outnumber boys with a similar condition 10 to 1, with nearly half of all African-American girls and approximately 15 percent of Caucasian girls showing clear signs of puberty, particularly breast development, by age nine. The timing of pubertal onset in humans is likely impacted by a suite of factors, including nutritional status, stress, socioeconomic status and genetics. But it has been hypothesized that exposure to estrogen-like compounds, including BPA, may partially explain this phenomenon.

Disruption of pubertal timing and the capacity to maintain a regular estrous cycle could result from developmental effects in the brain, the reproductive tract or both. Reproductive health ultimately depends on the proper organization and function of the hypothalamic-pituitary-gonadal (HPG) axis, a neuroendocrine system involving the hypothalamus; the pituitary gland, lying just beneath the brain; and the gonads (ovaries in females, testes in males). Hormones play a vital role in the sex-specific organization of all three components, so gestational exposure to endocrine disruptors has the potential to induce effects anywhere within the axis. Our laboratory’s work has focused primarily on the brain.

Chasing Meaningful Effects

Within the brain, volumes of sexually dimorphic nuclei in the hypothalamus are commonly used as a biomarker to study the neurodevelopmental impacts of endocrine disruptors. In the rodent brain, one of the most frequently studied regions is the sexually dimorphic nucleus of the preoptic area, known as the SDN, which is considerably larger in males than in females. The SDN is sensitive to steroid hormones from gestational day 18 until four days after birth. Following this critical period, a phase of sexually dimorphic apoptosis, or programmed cell death, begins, ending on postnatal day 12. Early exposure to estrogen, derived from the conversion of testicular androgens, protects the male SDN from apoptosis. In females, because estrogen levels are low during this period, apoptosis in the SDN progresses unabated, resulting in a smaller SDN.

Attempts to evaluate the impact of BPA exposure on SDN volume, in both males and females, have yielded largely negative and somewhat inconsistent results. For example, we determined that administration of the relatively high dose of 500 micrograms BPA over two days, beginning the day after birth, failed to alter SDN volume in male rats. Other studies, using higher and lower doses of BPA and exposure periods spanning both the gestational and postnatal periods, have also largely failed to find evidence for BPA effects on SDN volume.

Although these results are initially reassuring, we’ve concluded that using the SDN as a biomarker for endocrine disruption is problematic for several reasons. First, it appears to be relatively resistant to most endocrine disruptors, particularly in the Sprague Dawley rat. In addition, the functional role of the SDN is not readily apparent. It has long been accepted to play a role in male sexual behavior, but its role appears to be minimal. A final caveat is that humans have no clear SDN, so it is not entirely evident which reproductive health effects might be predicted by disruption of this region in rodents. It may be a useful region, however, to demonstrate mode of action. For example, we have found that BPA significantly increases the number of SDN cells containing calbindin-D28k, a protein thought to protect against apoptosis. This effect is consistent with the hypothesis that BPA acts as an estrogen mimic. Collectively these data show that, although the SDN is a popular marker for endocrine disruption, it may not actually be a very good one.

Another sexually dimorphic brain region that we and others have examined is the anteroventral periventricular nucleus (AVPV). In contrast to the SDN, the AVPV is larger in females than in males, and neonatal estrogen exposure induces, rather than prevents, the apoptosis that produces the size difference. Also, the functional significance of the AVPV is very well understood. The AVPV is essential for the coordination of the hormonal and environmental signals that regulate the secretion of gonadotropin releasing hormone (GnRH). In normal adult females, a “surge” of GnRH, elicited though positive feedback by the preovulatory rise of estradiol, induces the release of luteinizing hormone and, consequently, ovulation. In male rodents, neonatal estrogen, aromatized from testicular androgen, acts to defeminize the male AVPV such that GnRH neurons are no longer capable of responding to elevated estrogen levels with a surge of GnRH. Similarly, neonatal estrogen administration can defeminize the female AVPV, resulting in the inability to generate a GnRH surge in adulthood. The clearly defined functional significance of this region, combined with its sensitivity to estrogen during a discrete neonatal window, makes it a potentially ideal target for examining the impact of BPA exposure on sex-specific brain organization.

We found that administering the relatively high dose of 500 micrograms of BPA to male rats for only two days within this critical neonatal window of estrogen sensitivity did not affect the overall size of the AVPV. It did, however, result in a more female-typical number of dopaminergic neurons within the AVPV. Beverly Rubin of Tufts University subsequently reported finding male-typical numbers of dopaminergic neurons in the AVPV of female mice exposed from gestation through lactation to lower, environmentally relevant doses but did not look at AVPV volume. Together, these studies suggest that the AVPV is likely more sensitive to endocrine disruption than the SDN. They also support the hypothesis that BPA exposure may disrupt sexual differentiation of the brain in general, and within the AVPV specifically.

Another group of neurons of interest within the AVPV is the newly discovered kisspeptin neurons. It is now apparent that the kiss1 gene, which codes for a family of proteins called kisspeptins (KISS), is vital for the regulation of GnRH secretion in many species, including humans. It appears to be essential for the timing of both pubertal onset and ovulation. People with a mutated form of the kisspeptin receptor never enter puberty and remain hypogonadal throughout life. In the rat, there are two major populations of KISS neurons, one in the AVPV and one in the arcuate nucleus, another sexually dimorphic nucleus in the hypothalamus. The density of KISS neurons appears only to be sexually dimorphic in the AVPV, with females having significantly more KISS neurons than males. This population is thought to be critical for initiating the surge of GnRH that precedes ovulation.

To establish whether neonatal exposure to BPA could disrupt development of the AVPV KISS system, we injected rats with either 50 milligrams or 50 micrograms of BPA per kilogram daily for four days starting at birth. In rats, this is when the KISS system is most sensitive to estrogen. (In humans, this sensitivity window likely occurs in the early part of pregnancy’s second trimester.) In female rats, only the higher dose of BPA affected AVPV KISS levels. No effect was observed in the males. Interestingly, however, we also found a slight, but statistically significant, reduction of KISS in the arcuate nucleus of females exposed to the higher BPA dose. In contrast to the AVPV, the number of KISS neurons in the arcuate nucleus is not thought to be sexually dimorphic, and this population appears to be important for the regulation of steroid negative (rather than positive) feedback. Collectively these findings are important because they demonstrate that permanent, sex-specific effects within the brain are possible at exposure levels approximately equivalent to the LOAEL. By definition, no effects should be observable at or below this level.

Answering the Critics

Our work has been criticized because the animals in our studies were exposed by injection rather than by mouth. Humans are primarily exposed orally so there is concern that injection is not an appropriate exposure route for animal studies. This argument, although logical on its face, is not so straightforward. Injection bypasses metabolism, a process that conjugates most BPA and renders it inactive. The plastics industry has long argued that BPA is rapidly metabolized and excreted in humans, and is therefore not a threat. It has also been posited that, because non-oral administration, such as injection, bypasses metabolism, it presumably results in higher circulating levels of estrogenically active, or unconjugated, BPA. There are two significant problems with these assumptions. First, at least one study comparing oral administration and injection in neonatal rodents found no qualitative difference in the circulating levels of unconjugated BPA or its metabolites. Second, rodents and humans do not metabolize BPA the same way. In humans, the majority of the metabolites end up in the urine while in rats they are excreted in the feces. In addition, oral dosing is often stressful for animals, and does not adequately replicate exposure through dental sealants or implanted medical devices. Finally, even if most BPA is conjugated in adult humans, this may not be true for children or infants because their gastrointestinal systems are not fully mature. Considerable scientific debate persists regarding the capacity for immature rodents and humans to adequately metabolize BPA. (To see a timeline of responses to BPA, click the image at below.)

Our rodent brain research has also been challenged because of key anatomical differences between the rodent and human brains. Most significantly, in humans and other primates it appears that androgens, rather than estrogens, are primarily responsible for masculinizing the hypothalamus. Therefore, disruption of AVPV organization in rats, the argument goes, may not predict a concomitant effect in humans. Disruption within the rat AVPV does, however, clearly show that BPA has the potential to interact with developmental estrogen pathways, an effect that may impact aspects of human brain development. Another caveat is that humans have no AVPV. Humans do have kisspeptin neurons and discrete brain regions that coordinate GnRH release. So studying this neuronal population in rats will likely be informative for BPA’s effects on humans as well. These caveats again highlight the difficulties in translating results from rodents to humans but do not discount the importance of animal work by us or others. It is important to keep in mind that the developmental period in which steroid-sensitive organization occurs in humans happens largely during gestation rather than in the first few days of neonatal life. Thus, when exposing rats neonatally it is imperative to select BPA doses that correspond to prenatal exposure levels in humans.

It is clear that BPA exposure can disrupt pubertal timing and compromise the capacity to maintain a regular ovulatory cycle in rodents. It is likely that these defects result from the abnormal organization of the hypothalamic- pituitary-gonadal axis, the crucial neuroendocrine pathway that regulates reproductive function. Our work has found evidence for effects in the brain, but growing evidence suggests that the entire axis is vulnerable. For example, National Institutes of Health researchers have identified numerous reproductive-tract abnormalities in aged female mice exposed to BPA in utero at doses as low as 0.1 microgram per kilogram. We have observed cyst-like structures in rats neonatally exposed to 50 milligrams BPA per kilogram. Increased numbers of blood-filled ovarian bursae (indicative of advanced reproductive age), abnormal numbers of antral follicles, irregular chromosomes and decreased corpora lutea (tissues vital to a fertilized ovum) have also been observed by a number of research groups following exposure to BPA during development. BPA has also been found to induce apoptosis and cell arrest in cultured ovarian granulosa cells, suggesting that BPA may also impact the adult ovary. These studies indicate that the ovary may be a particularly sensitive target of BPA. Other laboratories have observed that gestational exposure to BPA at doses below the reference dose can result in numerous uterine abnormalities (including many that commonly precede carcinogenesis), alter mammary gland development, diminish the capacity to maintain pregnancy and induce abnormalities in the prostate.

It is also important to note that a handful of studies have found no effects from BPA at all. The most recent of these, published by EPA researchers in October of 2009, found no changes in the timing of pubertal onset, sexual behavior or fecundity among female rats after exposure to BPA during gestation and lactation. Inconsistent results continue to plague the field and make human risk assessment extraordinarily difficult. It is not readily clear why some effects are not easily replicated by other groups.

Resolving how to interpret evidence from laboratory animals, and soon, is pivotal because BPA is not the only endocrine disruptor people are exposed to. Thousands of other compounds are also suspected of having similar properties, including some plasticizers, flame- retardants, pesticides and anti-microbials. On top of that, it’s important to consider the combined effect of exposure to multiple endocrine disruptors. A research group at Washington State University recently reported that they could not replicate previously published BPA effects in the mouse ovary. They ultimately determined that effects were only observed in mice maintained on soy-rich diets, leading the authors to hypothesize that diet, along with methodological differences, could explain why the literature surrounding “low dose” effects of BPA is fragmented and inconsistent. The concept of mixture effects is an evolving area of endocrine-disruption research. It could have profound implications for human health if these compounds are found to be more likely to produce significant health effects collectively rather than individually.

Another sobering possibility is that endocrine disruptors could have transgenerational effects. For example, there is emerging concern that the children of DES daughters (referred to as DES granddaughters) might also develop reproductive problems. For these girls, their exposure occurred while they were only germ cells in their mothers’ developing ovaries, within the wombs of their grandmothers. This concern arose from laboratory data indicating that the offspring of female mice exposed in utero were more likely than unexposed control animals to develop reproductive-tract lesions. There are not enough human data to indicate a trend for deleterious effects in DES granddaughters. But this cohort is still quite young. Continued monitoring of these women as they age will be required.

The precise mechanisms through which endocrine-disrupting effects transmit to subsequent generations are not well understood, but emerging evidence indicates that epigenetic mechanisms might be primary. Epigenetic inheritance involves changes in gene expression patterns without changes in gene sequence. Such effects include DNA methylation and histone modifications. If epigenetic modifications occur within the germ cells, transmission to subsequent generations is possible. Randy Jirtle at Duke University found evidence in agouti mice that suggests that BPA has the potential to induce epigenetic effects. Other compounds, including polychlorinated biphenyls (PCBs) and the fungicide vinclozolin, have been shown to produce transgenerational effects, perhaps through epigenetic mechanisms. This newly discovered and evolving area of research has once again challenged toxicologists and introduced a novel method by which endocrine disruptors and other toxicants may affect vertebrate physiology and behavior.

How Much Evidence is Enough?

Given this complex context, a clear, science-based strategy for identifying, screening and regulating suspected endocrine-disrupting compounds is badly needed. Nearly 40 years after DDT, the first recognized endocrine disruptor, was banned, new chemicals in the U.S. market are not routinely screened or tested for endocrine- disrupting properties. Congress created the Endocrine Disruptor Screening and Testing Advisory Committee in 1996 to recommend how the EPA should test and screen these compounds, but progress has been frustratingly slow. A list of compounds to be screened was not compiled until April of 2009, and it included only 67 chemicals, a tiny fraction of the thousands now suspected of having endocrine-disrupting properties. It also has not yet been determined how the screening should be conducted and which biological endpoints should be used.

It remains unclear how much evidence is needed to evaluate health risks from compounds such as BPA. The NTP used 261 publications to conclude that there is “some concern” regarding BPA exposure. The FDA, using far fewer, has so far insisted there is little to no risk. Who is right? Most of the studies used by the NTP were conducted in university laboratories. In contrast, the FDA relied mostly on data provided by industry. This schism occurred because the NTP reviewed scores of studies by academic scientists. In contrast, the FDA relies most heavily on studies conducted using a set of guidelines called “good laboratory practices” (GLP). These guidelines specify how data must be organized and deal mostly with quality assurance, but they do not necessarily assure good study design, use of appropriate controls or robust statistical analysis. GLP studies are expensive. Thus they are nearly all conducted by private industry or contract labs, not academic labs. U.S. health agencies need to update their risk assessment strategies so that all of the data being generated and analyzed by agencies such as NIH, FDA and EPA are coordinated, shared and given equal scrutiny.

Although research to date is not conclusive, there certainly is sufficient evidence to warrant concern about potential long-term effects from BPA exposure. Only a handful of studies have looked at associations between BPA exposure and disease outcomes in humans, some of which have found correlations. It is not ethically sound to expose people to a suspect compound and watch what happens. Even if it were, it would take many years for our slow-to-mature species to display any effects from the encounters. Research in animals, however, is robust. It indicates that BPA may disrupt reproductive tract development, sex-specific neuroendocrine circuitry and fertility, even at doses considered relevant for humans. Since laboratory animals are used in other aspects of human health research, including drug development, it is reasonable that evidence obtained from them should play a central role in any comprehensive human-risk assessment for BPA. When combined with human epidemiological studies, cell-culture assays and high-throughput genomic studies, evidence from experimental animals is likely the best tool we have to make predictions about human risk. That is especially true for the long-term consequences of early exposures during critical developmental windows.

DDT: A Lesson From History

If rigorous scientific assessment doesn’t take the lead in deciding the fate of BPA, politics could, as happened with DDT regulation decades ago. That could open doors to long-term uncertainty and discord. The unintended environmental impacts of DDT were eloquently documented in Rachel Carson’s 1962 best selling book Silent Spring, which launched the modern environmental movement. Carson argued that by liberally spraying pesticides in our zealous determination to destroy “pests,” we risked the systemic destruction of our environment and ourselves. No scientific consensus on DDT’s true threat to people was reached, but in the summer of 1972 the EPA’s top administrator, William Ruckelshaus, announced a near ban of the compound anyway.

Nearly 40 years later, the DDT debate is not over. Most countries banned the agricultural use of DDT by the 1990s, but it is still used in many parts of the world to control mosquitoes, especially where malaria, a disease that kills more people than cancer, heart disease or AIDS, is endemic. Whether DDT causes disease or impairs reproductive development remains the subject of investigation and a controversial topic. Many scientists and policy makers are skeptical that it does; others are convinced.

Of course, DDT undoubtedly saved lives, and likely still does. No such case can be made for BPA. It is time to develop a clear and comprehensive strategy for assessing the potential public health consequences of endocrine disruptors such as BPA that may contribute only economic value. Failing to do so may put future generations at unnecessary risk. While the international debate over BPA’s safety continues, we continue to be exposed, not only to BPA, but also to the many compounds like it.