BPA

New analysis points to danger in FDA’s allowed levels of BPA in food

July 16, 2020 By Roger Meissen in Research Tags: BPA

Jiude Mao works on BPA testing in the lab of Cheryl Rosenfeld.

By Mariah Cox | Bond LSC

Bisphenol A, more commonly known as BPA, has been a source of scientific dispute for the past decade. With a lack of consensus among scientists, consumers are left unaware of the potential harms of the chemicals in plastic.

In response to a recent report by the Food and Drug Administration (FDA) that claims BPA is safe at the current levels occurring in foods, Bond Life Sciences Center principal investigator Cheryl Rosenfeld and a group of researchers across the country have teamed up to release a secondary analysis of the existing data, which disputes this claim.

The industrial chemical is used in manufacture of plastics and resins, and it is commonly found in plastic food containers, water bottles, food can linings and other consumer products. BPA can leach out into water supplies and food where humans and wildlife may be exposed to this ‘persistent chemical’ by ingestion or inhalation.

All of the researchers on the second report were a part of the original team put together by the FDA to study the effects of BPA. However, many researchers on that team disagree with the FDA’s re-analysis and interpretation of their individual findings.

By using the publicly available data published on the National Toxicology Program’s website, these scientists reevaluated the information originally compiled by Rosenfeld and dozens of colleagues as part of a Consortium Linking Academic and Regulatory Insights on Bisphenol A Toxicity (CLARITY-BPA).

Cheryl Rosenfeld had concerns of this Consortium project from the beginning.

“The idea at the outset was that individual investigators and FDA scientists partner together to address the question as to the safety of BPA, but even at the initial meetings, several concerns were raised,” Rosenfeld said.

The major source of disagreement boiled down to lab procedures, statistical analysis and a lack of regard for the inter-related effects of BPA on possibly multiple target organs and bodily functions. Going into it, the researchers had minimal input into the general experimental design, including a rat model that may be less sensitive to the effects of this chemical, the dosages of BPA that were tested, the fact that BPA was administered by what many consider a stressful procedure, oral gavage, and the period of administration.

One problem that was not thoroughly considered is the potential for nonmonotonic effects of BPA. That essentially means BPA shows adverse effects on the body at low and high doses, but not in between or middle-of-the-road doses.

On top of discrepancies over the research procedures, the researchers criticize the FDA for using stringent statistical analysis that may filter out important differences between groups.

“It’s like a metaphor about dropping your keys in a parking lot and looking over by the curb for them because there’s better light there,” said Gail Prins, a professor at the University of Illinois – Chicago and a collaborator on the original and secondary research project. “They’re concluding that BPA is not significant, but they’re not looking in the right places for significant results.”

In statistics, there are type one and type two errors. A type one error concludes that the results of the study were statistically significant when they’re not. Vice versa, a type two error concludes that the results are not statistically significant, but they are. Also, margin of error comes into play. P-value — a measure of deviation that determines which results are noteworthy — sets the stage for what is considered significant. Based on the method of a study, researchers can have stringent requirements for assessing the significance of a result (p≤.01), but most research uses p≤.05.

In simpler terms, p≤.05 allows researchers to be 95 percent certain that a result is meaningful. While the FDA used a p-value of <0.05, the researchers in the secondary study believe that the FDA failed to look at the statistical significance of the inter-related effects of BPA on multiple parts of the body, including the mammary glands, ovaries, kidneys, the prostate gland and cognitive-behavioral function.

Additionally, the statistical approaches the FDA sought to use would require hundreds of research replicates to be statistically valid. The FDA only had a budget to repeat the experiments up to 12 times per group, which some investigators questioned whether findings on these alone, especially with the methods the FDA sought to use, would provide meaningful results.

In 2012, the FDA banned the use of BPA in baby products, although that decision was largely due to public concern. However, the primary route of exposure to the effects of BPA are before babies are born. Since BPA is present in products used by pregnant mothers, it can lead to the development of health problems in babies including cancer later on in life.

The original statistical analysis for Rosenfeld’s portion of the project was done by Mark Ellersieck of MU, who has 30 years of experience, and a statistician with the FDA. When the analyses disagreed with each other, a neutral third-party was brought in to review the approaches used by Ellersieck and corroborated they were appropriate for the study design.

Now, Jiude Mao, a research scientist from the Division of Animal Sciences in Rosenfeld’s lab at Bond LSC, is working with Rosenfeld to reanalyze the results of the original study.

“I downloaded the raw data package online,” Mao said. “If you look at the effects of BPA on individual organs versus combining them and looking at its effects on multiple organs, the picture is very different.”

By using special informatics approaches, Mao found that the lowest dose of BPA tested simultaneously led to multiple effects on various target organs in females including the ovaries, uterus, mammary glands, heart, and fat tissue. In males, the prostate gland, along with the heart and adipose tissue showed inter-related changes due to BPA exposure.

Mao and Rosenfeld have also linked multi-organ effects of BPA at two other doses, with all doses tested currently considered safe by the FDA. They examined these inter-relationships at three age ranges: 21 days of age, 90-120 days of age, and 180 days of age. To the investigators’ knowledge this is the first type of toxicological study that has linked such data obtained in multiple investigators’ laboratories and shown such complex relationships.

The data from these three doses of BPA and three age ranges clearly indicate that BPA affects on a single organ can radiate out to affect many other organs throughout the body. By tugging on one organ, BPA can damage intricate webs that connects organs to each other. Such inter-relationships between individual CLARITY-BPA investigator data have not been considered by the FDA.

While a consensus hasn’t been met between the two parties, a potential solution for the data analysis discrepancy could be looking to machine learning or ‘deep learning’ to avoid human error or bias. This would include inputting both data sets into a program that can assess what the similarities and differences are and why the two groups are achieving different conclusions. This approach would ensure more confidence in the accuracy of the results instead of choosing a side to believe based on human calculations.

For the researchers, reevaluating the data means providing the full scope of the effects of BPA on multiple parts of the body. It also means giving consumers the correct information so that they can make well-informed decisions about their health.

“I am concerned that government agencies are not providing the public the fully story as to how BPA exposure might affect various organs, especially in infants exposed to this chemical during pre- and post-natal development when they do not have the full capacity to metabolize BPA and their organs are developing at this time,” Rosenfeld said.

Rosenfeld was joined by Jerrold Heindel, Scott Belcher, Jodi Flaws, Gail Prins, Shuk-Mei Ho, Juide Mao, Heather Patisaul, Ana Soto, Fred vom Saal and Thomas Zoeller from the Healthy Environmental and Endocrine Disruptor Strategies Commonweal, North Carolina State University, University of Illinois at Urbana-Champaign, University of Illinois at Chicago, University of Cincinnati College of Medicine, University of Missouri and University of Massachusetts at Amherst in this data reevaluation. Read more of their secondary results at the Journal of Reproductive Toxicology and see the original FDA CLARITY-BPA publication at FDA.gov.

Read more about this story at Environmental Health News. Cheryl Rosenfeld will be a featured speaker July 21 at 11 a.m. as part of a free webinar on this paper hosted by the Collaborative on Health and the Environment. Sign up at https://www.healthandenvironment.org/webinars/96533.

Making sense of junk

April 27, 2020 By Mariah Cox in Research Tags: BPA, endocrine disruptors, genistein, Junk RNA

Mary Butler, an undergraduate from Truman State University, gains experience working on experiments in the lab of Bond LSC’s Cheryl Rosenfeld. | photo by Roger Meissen, Bond LSC

By Mariah Cox

How did an undergraduate student from Truman State University spend last summer working on a research project with a Bond Life Sciences Center primary investigator and become on track to be published as first author several months thereafter?

A nationwide National Science Foundation (NSF) sponsored program has allowed Mary Butler to jump-start her research career early on.

Butler, a sophomore biochemistry and molecular biology student, wanted to get a head start on research to give her a leg up and figure out what her future might look like. As a freshman, Butler joined the Missouri Louis Stokes Alliance for Minority Participation (MOLSAMP) program and sought out opportunities that aligned with her science interests.

MOLSAMP is a collaborative effort among seven public universities, a private university and a community college. The alliance aims to increase the number of underrepresented minority students throughout the state of Missouri who pursue undergraduate degrees in science, technology, engineering or mathematics (STEM). The Missouri chapter is part of a greater program with the Louis Stokes Alliances for Minority Participation sponsored by the NSF, and Butler qualified because of her Mexican American background.

This past summer, Butler started traveling the 90 miles from Kirksville to Columbia to work with Cheryl Rosenfeld, a primary investigator at Bond LSC. Her research primarily focused on how genistein, a soy-derived phytoestrogen, and BPA, an industrial chemical, affects the behavior of mice.

“BPA and genistein are endocrine-disrupting chemicals, so they can mess with the hormones of an animal and potentially humans. I specifically looked at the different behaviors of California mice under four different diets,” Butler said.

The researchers administered a control, a genistein diet, a low dose BPA and a higher dose BPA diet to pregnant mice. After the baby mice were born, the researchers weened them from the mother to observe if the parents’ diet affected what the pups ate. Additionally, at 30 days, 90 days and 180 days, they put the pups through various social tests to see if their diet and their parents’ diet affected the brain and behavioral patterns.

They used socio-communication testing to determine whether developmental exposure to these chemicals led to deficits in these behaviors, which are reminiscent of those seen in children with autism spectrum disorder (ASD). The results point to less socialization among the mice exposed during the pre- and postnatal period to genistein or a low dosage of BPA.

Afterward, Butler measured gene expression changes in the hippocampus, which regulates learning and memory, and the hypothalamus, which guides socio-sexual behaviors. Besides examining for mRNA that encodes for proteins that can act within or outside of a cell, Butler and Rosenfeld also are the first group to examine whether developmental exposure to BPA and genistein affects the expression of so-called junk RNA within the brain.

Junk RNA strands are very short and do not give rise to proteins. For these reasons, they were historically dismissed as not important in cellular biology. However, it is increasingly apparent these RNA strands, also now called microRNA or miR for short, play vital roles in diverse cells throughout the body.

One way they act is to bind mRNA that would otherwise encode for proteins. In so doing, they help contribute to the demise of these mRNA strands. Essentially, the DNA can be transcribed to mRNA but miR binding prevents them from making a protein, acting as an epigenetic modifier that doesn’t alter the DNA or mRNA. In this sense, miR may serve as a system to help regulate mRNA.

While other research groups have examined how BPA affects miR patterns in the placenta and testes, no previous research group has done so in the brain, even though this organ is vulnerable to early exposure to BPA and genistein.

When Butler started in the Rosenfeld lab, she expressed a desire for challenging and novel work. With assistance from Jiude Mao in the Rosenfeld lab, they set out to test whether developmental exposure to BPA and genistein could alter miR decrease in ASD patients.

Notably, Butler’s work suggests that indeed such chemicals caused down-regulation of the same miR that are reduced in ASD patients. Normal expression of such miR appears to help prevent cell death, oxidative-stress damage, and yield other beneficial effects, and thus, reductions in these miR may leave California mice and, presumably, autism patients more vulnerable to such deleterious effects exposed to such chemicals.

Butler’s studies also suggest that changes in mRNA and miR profiles are associated with socialization deficiencies observed in California mice. Further testing is needed to confirm whether these molecular changes contribute to these behavioral disruptions, but, if so, it may pave way for new prevention and treatment strategies in those at risk of neurobehavioral disorders.

Since high school, Butler knew she wanted to pursue science. But she doesn’t quite know where to go from here with all the different options available; the opportunity at Bond LSC allowed her to look into one of those.

“I want to explore a lot of different types of research. My research with Dr. Rosenfeld is very different than the research I’ve been doing at Truman,” said Butler. “I had been looking at Dr. Rosenfeld’s research for a while and it looked really cool, so I sent her an email and asked if I could work in her lab over the summer. She said ‘yeah’ quickly, and it was really exciting.”

As a sophomore, Butler can say she’s the first author on a paper, and she knows how to do protein expression purification and polymerase chain reaction among other techniques.

“I really enjoy it because I’m more of a hands-on learner. Getting to do it hands-on, I’m able to absorb a lot more information,” Butler said. “In my classes, I’m able to follow along and talk about research things because I already know a lot of the techniques we discuss and practice in the lab.”

Butler’s research was published in the “Journal of Neuroendocrinology” in March 2020 and was funded by the National Institute of Environmental Health Sciences.

BPA research brings clarity to chemical’s influence on genes

July 23, 2018 By Roger Meissen in Research Tags: BPA, CLARITY-BPA consortium

By Roger Meissen | Bond LSC

After a decade of work, Cheryl Rosenfeld is no stranger to bisphenol A (BPA), and her most recent study challenges the dangers posed by developmental exposure the chemical.

Her results continue to raise concerns about how BPA can potentially turn on or off genes in animals and subsequent effects on that early exposure can have on the development and brains of rats. Their research was published in the journal Epigenetics in July.

Rosenfeld and the University of Missouri joined experts from University of Cincinnati and FDA researchers as part of the Consortium Linking Academic and Regulatory Insights on BPA Toxicity, or CLARITY-BPA Consortium project. This collaboration is one of several across the United States meant to judge the chemical’s effect using standardized protocols established by the FDA to determine whether BPA exposure, especially during perinatal life, leads harmful effects.

“This is the first study published since a February 2018 BPA statement that challenges the FDA assertion that there is no concern for BPA,” Rosenfeld said. “We’ve shown using the FDA models and studies done right there at their facility that, indeed, early life exposure to BPA can result in gene expression and epigenetic changes that persist into adulthood.”

The study looked at gene expression changes in two brain regions — the hippocampus and the hypothalamus. The hippocampus is associated with long-term learning and memory and the hypothalamus plays a large role in hormone production that influence both the endocrine and nervous systems and affects diverse behaviors, including socialization, sexual behaviors, and appetite control.

Partners at the FDA/National Center for Toxicological Research fed Sprague-Dawley groups of rats — a standardized animal model in this research — diets of BPA, the synthetic estrogen present in birth control pills, ethinyl estradiol, or a chemical-free diet during a developmental period.

The brains from these animals were sent to Rosenfeld’s laboratory, who took biopsies from specific regions of the brain. They used these samples to evaluate whether a group of 10 genes, shown to be affected by BPA exposure in other studies, was affected by this exposure. They also examined the DNA methylation patterns for the promoters of three of these genes to determine whether prior BPA exposure led to persistent epigenetic changes. Epigenetic modifications do not affect the DNA sequence itself but gene and/or eventual protein expression.

Investigators determined that for several of the genes examined BPA exposure altered the expression pattern relative to animals not exposed to either chemical. Sex differences in gene expression in these two brain regions exists in normal animals, and such differences might thus contribute to masculinization or feminization of the brain manifesting as differences in various behavioral patterns, such as male or female sexual behavior. However, previous exposure to BPA abolished many of these gene expression differences between males and females, suggesting that it could disrupt male- and female-typical behaviors. For a gene, brain derived neural factor (BDNF), involved in learning and memory, BPA exposure led to increased methylation of its promoter, which could affect the expression of this key gene. Hippocampal expression of several genes was associated with prior performance in a test designed to measure learning and memory.

“It has become increasingly apparent that BPA can act as a weak estrogen, but what we’re seeing in these results is that it can elicit other effects in addition to those mirroring estrogen and likely independent of estrogen receptor pathways,” Rosenfeld said.

Initiatives like this and other CLARITY-BPA studies aim to answer questions that may later inform government regulators on how to limit or balance the health effects of manufactured chemicals that end up in the environment and may affect human and animal growth in previously unknown ways. With more than 15 billion pounds of BPA were estimated to be produced in 2013, its ubiquitous use in making plastics, lining cans and other manufacturing is of concern. Rosenfeld hopes a closer look at its epigenetic effects may lead to better regulation of the chemical.

“When people are thinking about the effects of BPA, they need to be thinking about it on a molecular scale,” she said. “These results might be subtle, but they can lead to dramatic consequences with long-standing, irreversible changes. Once BPA exposure resculpts an animal’s brain through DNA methylation and other epigenetic changes, it may be permanent.”

“Gene Expression and DNA Methylation Changes in the Hypothalamus and Hippocampus of Adult Rats Developmentally Exposed to Bisphenol A or Ethinyl Estradiol: A CLARITY-BPA Consortium Study,” was published in Epigenetics in July 2018.

The Truth Behind BPA-Free Labels

November 2, 2017 By Samantha Kummerer in Research Tags: BPA

What companies aren’t telling you about their merchandise

By Samantha Kummerer | Bond LSC

Bisphenol A, otherwise known as BPA, is used to make plastic containers, coats the inside your metal food cans, and leaches into your food and water.

BPA has concerned scientists, health practitioners and the general public for many years because of its potential to mimic hormones and disrupt the developmental stages in animals.

Opposition to the chemical has led to certain manufacture’s marketing their items, such as food containers and water bottles, as“BPA–Free” and a global push to reduce the usage of BPA in commonly used household-items.

However, new evidence suggests consumers may not be as ‘free’ from harm as they think.

Bond Life Sciences Center researcher Cheryl Rosenfeld suggested that many consumers might not be aware of how these alternatives are made. BPA-substitutes — bisphenol S (BPS), bisphenol F (BPF) and bisphenol AF (BPAF) — aren’t too different from BPA chemically.

“They’re just playing with various synthetic structures is what many industrials groups are doing,” she said.

BPA is made up of two chemical compound groups called phenols. Picture two rings with different elements connected. In BPF, BPS and BPAF all those same parts are still present, and the only change is that the rings are rotated differently and contain various branching chemicals .

Since these alternate chemicals are structurally similar to BPA, they still bind to estrogen receptors, receptors to which the natural hormone estrogen binds and activates, which results in up- or down-regulation in the expression of various genes.

“The worst problem is that humans and animals are unknowingly serving as test subjects. Industrial companies are not required to show definitive evidence that these alternatives to BPA are safe. They can cite their own limited studies, but currently, no rigorous testing is required for these BPA-alternative chemicals that are being to flood the market. There is no requirement for the products that contain these chemicals to state as such. Thus, consumers cannot make educated decisions when purchasing various food and beverage products,” Rosenfeld said speaking of the industry.

Substitutes aren’t always better

Rosenfeld recently published a literature review exploring the use of the BPA-alternatives and their potential risk. She began the work out of curiosity since her Bond LSC lab studies the harmful effects of BPA.

After putting the puzzle pieces together, the big picture began to emerge that exposure of rodent models to these substitute chemicals exert analogous effects as BPA — depressive behaviors, increased anxiety, decrease in social behavior and decreased maternal/paternal care. In some cases, the alternatives have an even greater effect, according to Rosenfeld.

Understanding BPA

Production of the industrial chemical, BPA, began in the mid 1900’s to make plastics. Today, it can be found in everything from water bottles to storage containers and even food. With this increase exposure comes an increased risk to consumers.

Past studies linked exposure to the chemical with health effects on the brain and behavior and many other widespread effects.

When BPA enters the body, the can at least partially metabolize and break down the chemical via enzymes, which is then removed from the body. But there is a limited supply of such metabolizing enzymes used and those consuming BPA daily will end up overwhelming their body’s ability to metabolize and eliminate it with the net effect that BPA can accumulate over time and continue to exert potential harmful effects.

The most serious effect of BPA and now the substitutes happens during development. Fetuses have poorability to break down the chemical. Evidence links BPA exposure during development with neurological disorders like autism spectrum disorders (ASD) and attention deficit hyperactivity disorder (ADHD).

Most studies have examined the effects of BPA alternatives in rodents and zebrafish. While numerous factors prevent scientists from establishing causation in humans, Rosenfeld suggested it is time to at least start correlating early life exposure to BPA alternatives and risk of neurobehavioral disorders, including ASD, ADHD, and other neurodegenerative disorders.

“What’s worrisome is the women who are seeking to become pregnant or are currently pregnant, they are under the impression that they are making positive choices for their sons and daughters by using BPA-free products, but such products likely contain these BPA-substitute chemicals that can result in equal and possibly even greater negative consequences for their unborn offspring. Ultimately, the question is then what sort of products should be using?” Rosenfeld explained.

Humans aren’t alone in the impact. Rosenfeld explained these chemicals don’t break down in the environment and could play a part in the decline of certain animals. Previous studies by Rosenfeld’s lab and others at MU revealed BPA has the ability to feminize what should otherwise be male turtles.

“I just wish we could stop and pause and think about the havoc we are wrecking on our own environment and ourselves,” she said.

Without definitive results, a lot of unanswered questions remain. Future studies will likely explore the full range of effects caused by the BPA alternatives. Rosenfeld is calling for researchers to begin to treat these alternatives like BPA and put them through rigorous studies. These studies, however, come with a cost.

Rosenfeld’s lab is funded for BPA research. To add these additional chemicals to her current studies, the cost would double and triple due to the increased amount of animal test subjects that would be required.

“They keep upping the bar of how many replicas we have to test. If you want to show effects, if you want us to believe your results, we have to test 12-15 animals, well then you need to test another 12-15 animals in these groups and then your research dollars are all-of-a-sudden gone,” Rosenfeld said.

Research costs are going up and in the meantime BPA is still being produced in high quantities, so the industry as a whole has to decide where to allocate its funds.

“It’s a moving target. We’re trying to keep up and they keep synthesizing more and more chemicals everyday. The problem is we are completely outspent in terms of avaibable research dollars compared to the money industrial companies have on hand to fight tighter reguation. It’s not even a fair fight,” she said.

Rosenfeld hopes this new publication prompts scientists and the public to begin to question and call for more action on testing these pervasive BPA subsitutes. The silver lining is even though we can’t control our exposure to these chemicals, Rosenfeld hopes we might be able to find a way to combat our exposure.

Cheryl Rosenfeld is a Biomedical Sciences professor,a researcher in Bond Life Sciences Center, and research faculty member in the Thompson Center for Autism and Neurobehavioral Disorders. Rosenfeld specializes in how the early in utero environment can shape later offspring health, otherwise considered developmental origins of health and disease (DOHaD)She earned her bachelors and doctor of veterinary medicine degrees from the University of Illinois and her PhD from the University of Missouri.

BPA sex-reverses behavior in turtles

August 24, 2016 By Roger Meissen in Research Tags: BPA, turtles

Turtles could help determine how exposure to harmful chemicals during development affects male and female brains
Jeff Sossamon | MU News

Bisphenol A (BPA) is a chemical used in many consumer products including water bottles, metal food storage products and certain resins. Often, aquatic environments such as rivers and streams become reservoirs for BPA, affecting turtle habitats. Last year, a team of researchers led by the University of Missouri determined that BPA can disrupt sexual function in painted turtles, causing males to develop female sex organs. Now, the team has shown that BPA also can induce behavioral changes in turtles, reprogramming male turtle brains to show behavior common in females. Researchers worry this could lead to population declines in painted turtles.

“Previously, our research team found that BPA and ethinyl estradiol (EE2), a hormone found in birth control pills, could ‘sex-reverse’ turtles from males to females,” said Cheryl Rosenfeld, an associate professor of biomedical sciences in the MU College of Veterinary Medicine and an investigator in the Bond Life Sciences Center. “Painted turtles and other reptiles lack sex chromosomes. The gender of painted turtles and other reptiles is determined by the incubation temperature of the egg during development. Studies have shown that exposure to endocrine-disrupting chemicals (EDCs), such as BPA, can override incubation temperature and switch the sex of males to females. In our latest study, we found that BPA also affects how the male brain is ‘wired,’ potentially inducing males to show female type behavioral patterns.”

Researchers applied a liquid form of BPA and ethinyl estradiol to painted turtle eggs and incubated the eggs at a temperature that typically results in males. Five months after hatching, turtles were tested with a spatial navigation test that included four food containers, only one of which was baited with food. Each turtle was randomly assigned one food container that did not change over the trial period.

Researchers predicted that male turtles exposed to BPA and EE2 would exhibit improved navigational ability — similar to behaviors observed in female turtles. Results showed that developmental exposure to BPA and EE2 improved spatial navigational learning and memory in males, as evidenced by increased number of times spent in the correct target zone and greater likelihood of solving the maze compared to control turtles, who were male based on the lower incubation temperature.

“Previous studies have found that female turtles are much more adept at spatial navigation — think of female sea turtles that return many years later to the same beaches where they hatched to lay their own eggs,” Rosenfeld said. “We found that developmental exposure to BPA essentially overrides the brain development of male turtles as indicated by the enhanced navigational ability of the turtles we studied. While improved spatial navigation might be considered a good thing, it also may suggest that when they reach adulthood male turtles will not exhibit courtship behaviors needed to attract a mate and reproduce, which could result in dramatic population declines.”

Rosenfeld notes that this is the first study to show that these harmful chemicals not only reverse the physical sex-characteristics but also affect the brain in a turtle species. Turtles are known as an “indicator species” because they can be used as a barometer for the health of the entire ecosystem. By understanding the possible effects EDCs have on turtles, researchers might be able to understand the possible effects the chemicals have on other wildlife species and humans, Rosenfeld said.

“Effects of developmental exposure to bisphenol A and ethinyl estradiol on spatical navigational learning and memory in painted turtles (Chrysemys picta),” recently was published in the journal, Hormones and Behavior. Lindsey Manshack, a student at the time in Rosenfeld’s lab in MU’s Bond Life Sciences Center authored the study. Dawn Holliday, adjunct assistant professor of pathology and anatomical sciences in the MU School of Medicine and assistant professor of biology at Westminster College in Fulton, Mo., and Sharon Deem, director of the Saint Louis Zoo Institute for Conservation Medicine, contributed to the study. Funding was provided by Mizzou Advantage, the Office of Research and the Bond Life Sciences Center at the University of Missouri. The content is solely the responsibility of the authors and does not necessarily represent the official views of the funding agencies.

Maze Runners

October 20, 2015 By Roger Meissen in Research 16,588 Comments Tags: bisphenol A, Bond Life Sciences Center, BPA, CLARITY-BPA consortium, endocrine disruptors

Female rats struggle to find their way in BPA study from MU and the NCTR/FDA

Cheryl Rosenfeld is one of 12 researchers partnering with the NCTR/FDA to study BPA

Despite concerns about bisphenol A (BPA), academic and regulatory scientists have yet to reach a consensus on BPA’s safety.

The National Institute of Environmental Health Sciences (NIEHS), the National Toxicology Program (NTP), the Food and Drug Administration and independent university researchers are working together to change that.

Five years after the Consortium Linking Academic and Regulatory Insights on BPA Toxicity, or CLARITY-BPA for short, launched, results are beginning to come in. This new information will allow researchers to better compare the effects of fixed doses of BPA on the brain, various cognitive behaviors, reproduction and fertility, accumulation of fat tissue, heart disease, the immune system, and several types of cancers.

“The idea of this Consortium is to examine the potential systems that have been previously suggested to be affected by BPA,” said Cheryl Rosenfeld, an associate professor of biomedical sciences at the University of Missouri and one of twelve researchers involved in the project.

Rosenfeld’s group looked at spatial navigation learning and memory. They found that prenatal exposure to BPA could potentially hinder the ability of female rats to learn to find their way through a maze. This effect was not seen in male rats.

Approved by the FDA in the early 1960s, BPA can be found in a wide variety of products, including plastic food and drink containers with recycle codes 3 or 7, water and baby bottles, toys, the linings of metal cans and water pipes, even patient blood and urine samples.

BPA has structural similarities to estrogen and can potentially act as a weak estrogen in the body.

In Rosenfeld’s experiment, researchers at the National Center for Toxicology Research gave pregnant rats a fixed dose of BPA every day: a low, medium, or high dose.

After the baby rats were born, researchers continued to dose the babies, both male and female, according to what their mothers had received.

When these rats reached three months old, they were tested in a circular maze with twenty possible exit holes, one of which was designated as the correct escape hole. Every day for seven days, researchers tested the rats’ abilities to solve the maze in five minutes and timed them as they ran.

Rats solve mazes in three ways, Rosenfeld said.

They can run through the labyrinth in a spiral pattern, hugging the outer walls, and work their way in until they find the correct exit hole in what is called a serial search strategy.

Or they might move aimlessly in the maze using an indirect search strategy, Rosenfeld said. “In this case, the rats seemingly find the correct escape hole by random chance.”

Lastly, they can travel directly from the center of the maze to the correct escape hole. The third strategy is considered the most efficient method because the rats find their way swiftly, Rosenfeld said.

Sarah Johnson, a graduate student and first author on the paper, assessed each rat’s performance in the maze using a three-point tracking program that recognizes the rat’s nose, body, and tail.

Using the program, Johnson measured their performances in terms of the total distance traveled, the speed at which the rat ran the maze, how long it took the rats to solve the maze (latency), and how often the rat sniffed at an incorrect hole.

The last two parameters are considered the best gauges of spatial navigation learning and memory.

“What you expect to see is that they should start learning where that correct escape hole is,” Rosenfeld said. “Thus, their latency and sniffing incorrect holes should decrease over time.”

Rosenfeld’s group found that female rats that had been exposed to the highest dose of BPA since fetal development were less likely to find the escape hole than rats that hadn’t been exposed to BPA.

As for how this study may translate to people, Rosenfeld said, “the same brain regions control identical behaviors in rodents and humans.”

She considers it a starting point for setting up future experiments that take into consideration sex differences in cognitive behaviors and neurological responses to BPA.

Immediate next steps for the Rosenfeld group include analyzing tissue collected from the brains of rats that had undergone maze testing. Rosenfeld’s team of researchers will measure DNA methylation and RNA expression in the brain to determine which genes might be involved in navigational learning and memory. Their overarching goal is to determine how changes in observed sex- and dose-dependent behaviors occur on the molecular level.

NIEHS grant U01 ES020929 supported this research. Additional coauthors include Mark Ellersieck and Angela Javurek of the University of Missouri, Thomas H. Welsh Jr. of Texas A&M University, and Sherry Ferguson, Sherry Lewis, and Michelle Vanlandingham of the National Center of Toxicological Research/Food and Drug Administration. Read the full study on the Hormones and Behavior website and browse the supplementary data for this work.

Chemicals and couch potatoes

September 18, 2015 By Caleb O'Brien in Research Tags: BPA, Cheryl Rosenfeld, endocrine disruptors

Endocrine disruptors impact physical activity and metabolism in mice

By Caleb O’Brien | MU Bond Life Sciences Center

Could your experiences in the womb make you lazy as an adult?

A recent study of California mice suggests that early exposure to environmental chemicals can later impact an animal’s metabolism and level of voluntary physical activity, according to new University of Missouri research.

“We found that if we developmentally exposed California mice to bisphenol A (BPA) or ethinyl estradiol (EE), the estrogen present in birth control pills, it caused later disruptions in voluntary physical activity,” said Cheryl Rosenfeld, a researcher in MU’s Bond Life Sciences Center and associate professor of biomedical sciences in the College of Veterinary Medicine. “What that means is they move around less in their home cage, they’re more likely to sleep, and they engage in less voluntary physical activity.”

Rosenfeld’s lab studies the ways that exposure to environmental chemicals such as BPA can affect other behaviors, including cognition and parenting. Endocrine-disrupting chemicals can accumulate in the environment and act like the hormones naturally produced by many organisms, including humans. To test the chemicals’ impact on metabolism and activity, the lab used California mice. This mouse model is a good model for metabolic diseases. And because these animals are initially derived from the wild, they may better replicate the genetic diversity of most human populations.

The researchers exposed the mice to BPA and EE in the womb and until weaning via the mom’s diet. A third group of mice whose mothers were placed on a phytoestrogen-free control diet was not exposed to either chemical. The scientists then placed all the mice on this same control diet and measured their energy expenditure, body composition and level of voluntary physical activity as adults.

To test those attributes, Rosenfeld’s lab relied on a variety of tools and techniques. They rigged bicycle computers to “hamster wheels” to track how far, fast and for how long the mice ran. Using a device called a “Promethion continuous measurement indirect calorimetry system” the researchers continuously monitored the mice’s energy expenditure by measuring oxygen consumption and carbon dioxide production and by using a three-beam system, tracked the rodents’ movements during the dark and light cycles.

Later, the researcher measured the animals’ body composition using an EchoMRI, a tiny MRI machine the size of a filing cabinet, and finally measured circulating concentrations of glucose and hormones that regulate metabolism.

Female mice exposed to BPA and EE were less active than control mice. They moved around their cages less at night (when the nocturnal California mouse is considered most active), moved more slowly, drank less water, and spent more time sleeping. In addition, BPA-exposed females burned more carbohydrates relative to fats, as compared to control mice. This is similar to the difference between obese and slender humans, and many researchers believe that burning more carbohydrates relative to fats can lead to fats gradually accumulating in the body.

“It’s worrisome that environmental chemicals we are exposed to in utero can override our genes and disrupt our neuro-circuitry,” said Sarah Johnson, a research specialist and graduate student in Rosenfeld’s lab and primary author on the study. “The net effect is that we can have behavioral disruptions into adulthood, including altered physical activity.”

The researchers are currently conducting follow-up studies to determine if the changes caused by exposure to BPA and EE predispose mice to obesity and other metabolic disorders. They also are interested in exploring if exposure could affect the children and grandchildren of these mice and examining the potential underlying neural mechanisms.

“Our findings are significant because decreased voluntary physical activity, or lack of exercise, can predispose animals or humans to cardiovascular diseases, metabolic disorders and even cancer,” Rosenfeld said.

Other authors on the study are Angela Javurek and Michele Painter (MU Biomedical Sciences), Mark Ellersieck (MU Agriculture Experimental Station- Statistics), Charles Wiedmeyer (MU Veterinary Medical Diagnostic Laboratory and Department of Veterinary Pathobiology) and John Thyfault (Kansas University Medical Center, Molecular and Integrative Physiology)

The study, “Sex-Dependent Effects of Developmental Exposure to Bisphenol A and Ethinyl Estradiol on Metabolic Parameters and Voluntary Physical Activity” was supported by NIH Grant 5R21ES023150 (to C.S.R.) and R01DK088940 (JPT) and was published in the Journal of Developmental Origins of Health and Disease.

Poor parenting or BPA?

June 3, 2015 By Roger Meissen in Research Tags: bisphenol A, Bond LSC, BPA, endocrine disruptors

Endocrine disruptors alter parent behavior in California mice

California mice exposed to bisphenol A (BPA) or ethinyl estradiol changed their parenting behavior, according to an MU Bond LSC study. | Photo by Roger Meissen, Bond LSC

By Roger Meissen | MU Bond Life Sciences Center

What if a chemical changes the way an animal parents?

That could happen due to endocrine disruptors like bisphenol A (BPA).

A recent study of California mice exposed to BPA showed parents spend less time feeding, grooming and interacting with their babies, according to University of Missouri research. Even mother mice not exposed to the chemical parented differently if their male partner was exposed during development.

Most studies only use laboratory mice and rats — where the mother is the sole parental provider — so how early contact to BPA may affect the father and his partner remained a critical gap in existing research.

“The nature and extent of care received by an infant is important because it can affect social, emotional and cognitive development,” said Cheryl Rosenfeld, a researcher in MU’s Bond Life Sciences Center and associate professor of biomedical sciences in the College of Veterinary Medicine. “We found that females who were exposed early on to BPA spent less time nursing, so the pups likely did not receive the normal health benefits ascribed to nursing. Likewise, we found that developmental exposure of males and females resulted in them spending more time out of the nest and away from their pups, further suggesting that biparental care was reduced.”

BPA and other endocrine disrupting chemicals like ethinyl estradiol (EE) — found in birth control — concern scientists because they build up in the environment and mimic natural hormones produced by animals, including humans. Everyday exposure to these chemicals can impact offspring development and now have been found to alter adult behavior in test animals.

California mice have special significance for studying parental behavior. Unlike most lab mice, Californian mice pair up to mate and care for offspring. This monogamous behavior could give researchers insight into child rearing behavior found in most human societies and other biparental animals that would be impossible to measure in lab mice and rats.

MU graduate student and primary author Sarah Johnson worked with Rosenfeld to design the study to look at both sexes. Female and male mice were fed one of three diets — food supplemented with BPA or ethinyl estradiol or endocrine-free (control) food — two weeks before breeding. The mice were then randomly paired with the same mate for the entire study. The behavior of both sexes was then tracked for activities like time spent grooming pups, time spent in the nest and time mothers spent nursing.

But how do you measure the behavior of parents?

Rosenfeld’s team depended on hundreds of hours of video footage, taken at particular times of day and night for seven days, starting two days prior to birth. By using infrared cameras they tracked all 56 litters of mice, logging the number of and duration of activities mothers and fathers completed. During this time, the body weight and temperature of the F2 pups, who were not directly or fetally exposed to any chemicals, was logged to monitor their development.

While results showed reduced pup attention from BPA/EE exposed mother mice, the most intriguing result showed that unexposed moms mated with exposed fathers reduced the time they groomed and cared for offspring.

“These female mice have not been exposed here, but if you can see they are still reducing parental care when paired with the BPA/EE-exposed males,” Rosenfeld said. “And what’s even more interesting is that if a mother and father are both exposed, that parental care diminishes further, and becomes even more statistically significant.”

Researchers hope these results will spur others to look at long-term effects of endocrine disruptors on parenting behavior from generation to generation in animal models and, more importantly, in humans, to see if these chemicals can disrupt parental behavior of mothers and fathers, and if so, whether these effects can be transmitted to subsequent generations.

The study, “Disruption of Parenting Behaviors in California Mice, a Monogomous Rodent Species, by Endocrine Disrupting Chemicals,” was funded by the National Institutes of Health (Grant: 5R21ES023150-02) and was published in the journal PLoS One.

BPA overrides temperature to decide turtle sex

April 16, 2015 By Roger Meissen in Research Tags: Bond Life Sciences Center, BPA, Cheryl Rosenfeld, mizzou, turtles

The environmental build-up of bisphenol A (BPA) can result in a life-changing shift for aquatic animals.

For painted turtles, exposure to this chemical can disrupt sexual differentiation, according to new research in General and Comparative Endocrinology.

Scientists at the University of Missouri have teamed up to show how low levels of certain endocrine disruptors like BPA can cause males to possess female gonadal structures in newly-hatched turtles. This collaboration between MU, Westminster College, the U.S. Geological Survey (USGS) and the Saint Louis Zoo exposed turtle eggs to levels of BPA similar to those currently found in the environment.

“It’s important because this is one of the first times we’ve seen low doses of BPA causing disorganization or reorganization of the male gonad to resemble females,” said Dawn Holliday, adjunct assistant professor of pathology & anatomical sciences at MU’s School of Medicine and assistant professor of biology at Westminster College. “We’re not sure what this means in terms of population-level effects, but certainly it can cause some reproductive dysfunction for turtles.”

Painted turtle eggs were brought from a hatchery in Louisiana, candled to ensure embryo viability and then incubated at male-permissive temperatures in a bed of vermiculite. Those exposed to BPA developed deformities to testes that held female characteristics. Photo by Roger Meissen | © 2015 - MU Bond Life Sciences Center — Painted turtle eggs were brought from a hatchery in Louisiana, candled to ensure embryo viability and then incubated at male-permissive temperatures in a bed of vermiculite. Those exposed to BPA developed deformities to testes that held female characteristics.
Photo by Roger Meissen | © 2015 – MU Bond Life Sciences Center

Endocrine disruptors leach into rivers and streams and concern scientists because of potential effects on animals and humans. While BPA is used as a hardening agent in plastics, it also is used to line cans and in manufacturing where more than 15 billion tons are produced each year.

In the case of painted turtles, these chemicals have potential to alter sex ratios, which are normally regulated by temperature during incubation. Eggs exposed to cooler temperatures normally produce males and those hatched at warmer temperatures produce females.

Turtle eggs incubated at cooler temperatures result in male hatchlings while warmer temperatures cause females. Researchers are measuring the temperature and weight of this turtle. Photo by Roger Meissen | © 2015 - MU Bond Life Sciences Center — Turtle eggs incubated at cooler temperatures result in male hatchlings while warmer temperatures cause females. Researchers are measuring the temperature and weight of this turtle. Photo by Roger Meissen | © 2015 – MU Bond Life Sciences Center

In this experiment, turtle eggs were incubated at temperatures known to rear males and dosed with low, medium and high levels of BPA. BPA-exposed turtles were compared to hatchlings not exposed to chemicals as well as a group exposed to high levels of ethinyl estradiol — an endocrine disruptor found in birth control — at the USGS Columbia Environmental Research Center.

These doses resulted in turtle sex organs that should have been male , but abnormally contained female gonadal elements. The low dose represented BPA concentrations found in fields where turtles can nest while the mid and high doses approximate BPA levels near contaminated sites like landfills.

“We exposed the eggs for a limited amount of time right when they were most vulnerable to the effects,” said Cheryl Rosenfeld, a researcher at MU’s Bond Life Sciences Center and an associate professor of biomedical sciences in the College of Veterinary Medicine. “We found that we got partial feminization in more than 30 percent of turtle eggs exposed to BPA despite being incubated at male-permissive temperatures.”

Dawn Holliday (left), Caitlin Jandegian and Cheryl Rosenfeld examine turtle gonadal tissue to determine if BPA affected proper sexual development. Photo by Roger Meissen | © 2015 - MU Bond Life Sciences Center — Dawn Holliday (left), Caitlin Jandegian and Cheryl Rosenfeld examine turtle gonadal tissue to determine if BPA affected proper sexual development. Photo by Roger Meissen | © 2015 – MU Bond Life Sciences Center

These results give the team a look into what real-world exposure levels might mean in the wild and a starting point for comparison.

“Turtles are the most endangered vertebrate taxa and they have all sorts of conservation issues coming at them from people harvesting them to disease, and endocrine disruptors are another potentially big whammy they have against their conservation status,” said Sharon Deem, director of the Saint Louis Zoo’s Institute for Conservation Medicine. “This research is a stepping stone, and we are hoping we can apply these results to populations of turtles throughout the state and use these results as a marker to look at endocrine disruptors in the wild.”

Future studies plan to look at the underlying mechanisms behind sexual disruption and will extend the study to animals including fish and mammals. Rosenfeld’s laboratory is in the process of examining how early exposure of turtles to endocrine disruptors might affect cognitive behaviors, including spatial navigation ability.

Fred vom Saal, Curators Professor of Biological Sciences in the College of Arts and Science at MU, Don Tillitt, an adjunct professor of biological sciences at MU and a research toxicologist with the USGS, Ramji Bhandari, an assistant research professor of biological sciences and a visiting scientist with the USGS at MU and Caitlin Jandegian, a senior research technician at MU, all collaborated on the study.

Candling helps determine whether the painted turtle embryo is viable for the experiment. Photo by Roger Meissen | © 2015 - MU Bond Life Sciences Center — Candling helps determine whether the painted turtle embryo is viable for the experiment. Photo by Roger Meissen | © 2015 – MU Bond Life Sciences Center

Funding was provided by Mizzou Advantage, an MU initiative that fosters interdisciplinary collaboration among faculty, staff, students and external partners to solve real-world problems in four areas of strength identified at the University of Missouri. These areas include Food for the Future, Sustainable Energy, Media for the Future and One Health/One Medicine.

Canary in the water: The impact of BPA and estrogen on aquatic life

August 21, 2013 By Roger Meissen in Research Tags: BPA, Cheryl Rosenfeld, Painted Turtles

MU Bond Life Sciences Center investigator Cheryl Rosenfeld is studying the impact of BPA on painted turtles in collaboration with scientists from MU, the St. Louis Zoo, the U.S. Geological Survey and Westminster College. These turtles and many amphibians are extremely sensitive to environmental contaminants and may indicate wider issues from certain types of environmental contaminants.

Bisphenol-A (BPA) and other estrogenic compounds are becoming increasingly prevalent in the environment. More than 8 billion tons of BPA are produced each year in manufacturing, and pharmaceutical compounds like ethinyl estradiol make their way into rivers and streams. They can affect the sexual and cognitive development of animals.

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