As we described in our previous two posts, the EPA recently announced 23 chemicals that it will assess for health risks in 2013, including 20 flame retardants. One of the three chemicals that is not a flame retardant, 1-bromopropane (also called n-propyl bromide or nPB) is another brominated organic compound… that also prevents nerves from signaling properly.

nPB is used in industrial and commercial applications – by auto body shops, dry cleaners, and electronics manufacturers. It’s also used as a glue to hold together furniture cushions, the very same furniture cushions that are frequently embedded with the brominated and chlorinated flame retardants we study.

The New York Times recently highlighted the health risks, especially nerve damage, that furniture makers suffer when they breathe in nPB over a long period of time. Furniture-making workers who spray this glue can become permanently disabled, damaging their health, their happiness, and their ability to secure a stable income.

Available research on nBP is limited but clearly indicates that exposed mammals can have slowed nerve function and weakened limbs, especially in hind limbs, consistent with the foot and leg problems furniture workers report. nBP seems to damage the myelin sheaths that coat nerves. Myelin sheaths insulate the nerve and help contain the nerve signal, speeding its transmission. By damaging myelin, nBP allows the nerve’s signal to dissipate and slow.

This story makes me think about two things:

1) We as a culture need to better understand long-term risks. The New York Times article argues that the Occupational Safety and Health Administration (OSHA) focuses too much on short-term safety (e.g., dangers from ladders and stairs) at the expense of important long-term safety concerns, such as exposure to thousands of chemicals in the workplace. People tend to ignore far-off future risks, especially ones that slowly accumulate, for short-term benefit. We need to culturally counter that tendency.

2) With flame retardants, we’ve seen a slow cycle of compounds being taken out of use because of health concerns only to be replaced by new, unstudied compounds that are also unsafe. Somewhat similarly, furniture manufacturers landed on nPB glue to replace previous chemicals that harmed the ozone layer or human health.

We need a better system.  The Toxic Substances Control Act of 1976 (TSCA) does not require safety testing on all new chemicals put into use; EPA must instead determine that safety testing is needed. As ecotoxicologists, we don’t want to just describe how people and the environment can be harmed after the fact: We hope to prevent such harm in the future. Because of our federal funding, we don’t plan to endorse any specific bills for TSCA reform. However, we strongly recommend that new laws require demonstrated safety records for chemicals to be used industrially or commercially.

However, flame retardants are the subject of increasing scrutiny. One type of flame retardants—PBDEs—are known toxins, and are currently being phased out in the United States. The Duke Superfund Research Center’s own Heather Stapleton testified before the US Senate Committee’s hearing on the Toxic Substances Control Act (TSCA) about her research on PBDEs and evidence of their detrimental effects on human health.

Why is interest in understanding the effects of flame retardants on the rise? New types of flame retardants are replacing PBDEs, and little is known about their effects on human health to date. Many of the reasons scientists are concerned about flame retardants are similar to the reasons that DDT was banned in the US during the 1970s.

Flame retardants are ubiquitous. They are used in furniture, building materials, electronics, and baby products. Like DDT, flame retardants are persistent—they stay in the environment for a long time without breaking down into safer compounds, and they accumulate in our bodies. Due to their wide use for over 30 years, flame retardants have made their way into everything from dryer lint to our food to human blood and breast milk.

Couple all of this with known neurological and developmental effects of certain flame retardants. PBDEs, which are among the most well-studied flame retardants, have been linked to cancer. PBDEs can also impact on children’s health and development—such as low birth weight and IQ.

In our previous post, we mentioned 4 types of flame retardants on which the EPA will be able to conduct a full risk assessment under TSCA. These chemicals are TBB, TBPH, TCEP, and HBCD. We’ve compiled a list of how these chemicals are used and in which products below:

TBB (2-Ethylhexyl ester 2,3,4,5- tetrabromobenzoate)

• Used in: PVC, neoprene, coated fabrics, wall coverings, adhesives

TBPH (1,2- Ethylhexyl 3,4,5,6-tetrabromo-benzenedicarboxylate or (2-ethylhexyl)-3,4,5,6 tetrabromophthalate)

• Used in: polyurethane foam (in furniture and construction), baby products, wire and cable insulation, film and sheeting, carpet backing, coated fabrics, wall coverings, adhesives

TCEP (Tris(2-chloroethyl) phosphate)

• Used in: PVC, home electronics (including televisions and computers), adhesives, upholstery, carpet backings, rubber, plastics, paints and varnishes

HBCD (Hexabromocyclododecane)

• Used in: all polystyrene foam used in building insulation in the US, textile backcoatings for institutional products, present in some audio-visual equipment, refrigerator linings, and wire and cable coatings

HBCD is emerging as a particular chemical of concern because its use in insulation makes it popular among builders to improve energy efficiency in green homes. In addition, HBCD has been found in human breast milk and food.

The bottom line is we don’t understand how flame retardants affect our health, which is why the EPA’s risk assessments will provide crucial information on these pervasive chemicals. (At the Duke SRC, Project 2 is studying how chemicals, including flame retardants, impact thyroid hormones and the Neural and Behavioral Toxicity Assessment Core will be looking at how they affect neuro-development and behavior.) The EPA’s risk assessment will provide a clearer understanding of the human health effects of flame retardants. Stay tuned!

In a press release a week and a half ago, the U.S. Environmental Protection Agency announced that in 2013 it will look at the potential human health and environmental effects of 23 commonly used chemicals. This announcement is part of EPA’s work plan under the Toxic Substances Control Act (TSCA), an act passed in 1976 which grants EPA the “authority to require reporting, record-keeping, and testing requirements, and restrictions relating to chemical substances and/or mixtures.”

Which chemicals?

20 are flame retardants. Of the 20 flame retardants, 4 currently have enough data for the EPA to carry out full risk assessments to fully identify potential risks. They are

1. 2-Ethylhexyl ester 2,3,4,5- tetrabromobenzoate (TBB),
2. 1,2- Ethylhexyl 3,4,5,6-tetrabromo-benzenedicarboxylate or (2-ethylhexyl)-3,4,5,6 tetrabromophthalate (TBPH),
3. Tris(2-chloroethyl) phosphate (TCEP), and
4. Hexabromocyclododecane (HBCD).

The information from the full risk assessments from these 4 flame retardants will be used to understand the possible risks of 8 of the remaining flame retardants whose chemical structures are similar to the 4 above.

3 are non-flame retardants. EPA will begin the process of developing risk assessments for:

1. Octamethylcyclotetrasiloxane (D4),
2. 1-Bromopropane, and
3. 1,4 Dioxane.

EPA is asking the public, including chemical manufacturers, to voluntarily provide information on the toxicity of the chemicals to assist in their risk assessment and fill in data gaps.

For more information on EPA’s announcement, check out the links below.

EPA’s press release! The press release contains links to information on TSCA and it’s related work plans.

Mother Jones “EPA to Study Flame Retardant Chemicals. Finally.”

Chemical & Engineering News, American Chemical Society “Flame Retardants Under Scrutiny”

Interested in gaining some research experience with Duke’s Superfund Center? We have opportunities for you! Through our Research Experience for Undergraduates (REU) Program, you can apply to work with any of our 4 projects and 3 of our cores (Analytical Chemistry Core, Neural and Behavioral Toxicity Assessment Core, and Research Translation Core).

Contrary to the name of the program, you don’t need to be an undergraduate student. This opportunity is open to undergraduate and Master’s students.

Visit this page to learn more about the research opportunities available and how to apply. The deadline for applications is March 15, 2013.

For a pre-med student and biology major, my choice to research at Duke’s Nicholas School of the Environment this summer may seem out of place. Why not research at a cancer genetics lab or neuroscience lab? Well, about a year and a half ago, I took Joel Meyer’s introductory environmental science course and learned he was the PI (principal investigator) of a toxicology lab. I was interested in working there so I learned more…

According to Wikipedia (I know, land of all potentially false information), toxicology is “a branch of biology, chemistry, and medicine concerned with the study of the adverse effects of chemicals on living organisms”. If you look at Joel’s research interests online, they are described as “understanding environmental and genetic influences on organism health, with a focus on DNA integrity and oxidative stress.” His work sounded like a perfect mix of biology, environmental science (a passion of mine and, eventually, my minor), and health to me, so I expressed interest. By the end of my meeting with him, I knew I would be working with C. elegans (a nematode, or roundworm, about 1mm in length) and mitochondrial DNA, was emailed a few papers, and that was about it.

Exactly a week before my first day of work, I was startled to find Joel had emailed me to say he thought I would be working with John Rooney, a graduate student in the lab, and to look for him on my first day. I knew PIs weren’t very involved in their labs, but knowing Joel personally, I was surprised to find how little I actually communicated with him, and how many things he was responsible for. I later realized that even the limited communication I have with Joel is quite a lot compared to other labs.

I eventually found John the next week, and when we met, I figured he was a fairly typical grad student, most likely a few years older than me. I soon discovered that John was actually older than I thought (almost ancient, really—just kidding John!), because he had worked in a lab for several years after graduating college. I became very grateful for working with him, not only because he had so much experience, but also because he ended up being an incredibly patient and relaxed mentor.

At first, I started with learning basic tasks, like transferring worms or doing “egg preps”. John and I also talked a lot in the first few weeks, as he answered my many questions about the science involved in an experiment or a new lab technique. Because I was in the lab 40 hours a week, I was able to learn much more than if I had started during the school year, and after only a few weeks in the lab, I felt dramatically different from when I had first started. It made me think—if even a few weeks seem to make a difference, a year, or two, or three, or more, must make an enormous difference!

Shortly after learning some basic tasks, John started working with me on some behavioral assays. We “dosed” the worms with UV-C rays to mimic the DNA damage done by PAHs (polyaromatic hydrocarbons), which are compounds we focus on in our lab. After hatching, the worms are exposed to UV-C rays at 24-hour intervals for three days. Because nuclear DNA has a robust repair system, this 3-day dosing scheme allows us to assume nuclear DNA damage in the worms is minimal, while the mitochondrial DNA damage has accumulated. We know this exposure results in neurodegeneration, reduced ATP levels, and reduced movement; what we wanted to see is if the reduced movement is from the aforementioned neurodegeneration or reduced ATP levels (metabolic disorder), so we did four different assays testing chemosensation, osmotic avoidance, mechanosensation, and locomotion. Unfortunately, the behavioral assays were very difficult to interpret. Not only did we encounter a variety of problems with each individual experiment, but overall the assays relied on the movement of the worms, which, as I mentioned, was reduced through the dosing.

Eventually, after weeks of tweaked methods and unsatisfactory results, we decided to dose the worms with 6-hydroxydopamine (6-OHDA), which is a well-known positive control for neurodegeneration. We were looking for a difference in movement on foodless plates and normal food plates. Normal, wild-type worms should slow down on plates with food as they eat, and speed up on foodless plates as they search for food; this is referred to as a “slowing on food” response. As this response is dopaminergic neuron-dependent, 6-OHDA dosing is a good control for the assay—if the dosed worms were affected in the neurons related to the movement phenotype, they may not show the “normal” difference in phenotype. However, we saw that the 6-OHDA-dosed worms were perfectly fine, and displayed dramatic differences in movement between food and foodless plates. As 6-OHDA should certainly damage the worms’ dopaminergic neurons, we used this information to determine that neurodegeneration from the UV-C exposure would be near-impossible to detect through an altered movement phenotype if there wasn’t even a clear difference in the 6-OHDA-dosed worms.

Amidst that chaos, we’ve been doing a few other experiments as well. We started a fairly simple but tedious reproductive assay where we count brood size of the worms, and look at differences across UV-C exposure dosage. Essentially, we count how many babies individual worms lay. This could tell us the effect that PAHs may have on reproduction and development. We’re about to start this experiment for the third or fourth time. The first time, the dosed worms laid literally zero eggs, and the third time (recently), half the adults were dead so we have to start over. We may also look at the relationship between cep-1, or the C. elegans analog of p53, a tumor suppressor protein (and therefore cancer-preventing gene) and mitochondrial DNA damage signaling soon. The implications of this one are pretty clear, and I’m excited about starting it!

I’ve always heard about the long timelines in a research environment, but of course, I didn’t really experience it until I entered a lab myself. Although I was honestly relieved to stop the behavioral assays, I had to come to terms with the fact that I probably wasted a few weeks—and yet, there was no way of knowing this ahead of time. Such is science research! And even when I may have found slightly satisfying results, repetition, repetition, repetition, is key (you see what I did there), and an interesting finding is only the tip of the iceberg.

So this summer has been quite the learning experience for me. On top of my first research experience, I’ve been very fortunate to join a lab of friendly, helpful, and enthusiastic people. Oftentimes, we hear that lab experiences are shaped by who we work with. I’m very grateful to have a PI who fosters community within the lab, as well as a mentor willing to spend time to help me (and accept all my mistakes nonchalantly), while also giving me freedom and trust.

In addition to being introduced to a wonderful group of people, I feel I’m enjoying my experience in a way that contributes to several of my interests. Mitochondrial DNA is linked to several rare diseases and, as it is highly affected by toxicant exposure, is becoming increasingly important in toxicology. My interest in the connection between human and environmental health is now more than an abstract interest; I’m finally taking a small, but active role in exploring this connection. By exploring my various interests through the lens of toxicology, I’ve gained an enriching perspective on the fundamental link between them all, and I’m sure the future will only be more exciting!

Nick Tippens – Project 2, Dr. Stapleton’s lab

Amy Lin - Neural and Behavioral Toxicity Assessment Core, Dr. Levin’s lab

Akshay Save - Analytical Chemistry Core, Dr. Ferguson’s lab

Claire Carson – Research Translation Core

Please click the link below to open the document containing the descriptions for each of the opportunities. 

Summer 2013 Research Experience

For more information about the internships, please contact Dr. Ed Levin by email (edlevin@duke.edu).

When I began my summer work in Dr. Heather Stapleton’s environmental chemistry lab, I didn’t know exactly what to expect. I was told I would be hired as a member the Superfund Trainee Program, and that I would be working primarily with two of the lab’s Ph.D. candidates on their research with zebrafish. I actually worked next door to the Stapleton lab in the summer of 2011 for the Duke Wetland Center, handling general lab duties ranging from sample preparation to glassware cleaning. While it was a good experience, I was not consistently given what I considered truly impactful responsibilities. In contrast, last summer, by my second or third week in the Stapleton lab, I was spending my afternoons screening plates of zebrafish embryos dosed with various chemicals all on my own. It was rewarding to know that I lent a genuinely helpful hand to Dr. Stapleton’s research and the Superfund program at large.

As the weeks went on, I accumulated a basic (though still admittedly limited) understanding of the research for which I am assisting Dr. Stapleton’s Ph.D. candidates. The overarching focus of much of Dr. Stapleton’s research is investigating the human and environmental impact of chemicals used as flame retardants in consumer products as these chemicals leach out of those products over time. Many of these flame retardant chemicals are halogenated organic compounds, meaning that they are carbon-based compounds containing at least one atom of bromine, chlorine, or fluorine in their chemical structure.  As part of Superfund Project 2, some of Dr. Stapleton’s Ph.D. students are specifically exploring how certain halogenated organic compounds used as flame retardants impact thyroid function. The chemical structures of some of these compounds and of the compounds formed when they are metabolized inside an organism are similar to the chemical structures of some thyroid hormones. Due to these structural similarities, it is suspected that the presence of these toxic chemicals may interfere with normal hormone activity, particularly when an organism is in the earlier stages of development.

Because zebrafish are relatively inexpensive, develop extremely quickly, have transparent embryos, and have had their entire genome sequenced, they provide a great model for testing the developmental effects of halogenated organic chemicals on the thyroid system and on the organism as a whole. Therefore, much of my time as a Superfund trainee was spent working with zebrafish. I learned how to feed the fish, how to breed the fish, how to harvest embryos, how to select good embryos for exposing to chemicals, how to expose the embryos properly, how to screen the embryos for defects, and how to properly euthanize the embryos when experiments end. I developed skills more quickly than I anticipated, but I still found myself with endless questions and at times only a basic understanding of what’s really going on with the science behind all of the research.

Last summer I was exposed to just a small sample of the simply incredible amount of time and effort necessary to carry out successful research. I saw how maddeningly frustrating the world of science can be (it can take weeks or months to get crucial analytical machinery fixed in a lab), as well as how rewarding it can be (just this week the Environmental Protection Agency has decided to further investigate flame retardants due to the weight of research suggesting their harmful effects – research that members of the very lab in which I’m working have significantly contributed to). I was around people who are passionate about what they are researching and excited about what they may discover, as well as people who are burned out and tired of the grind and repetition of science. It was a challenging learning experience for me as a Superfund Trainee, and ultimately I don’t think I will pursue a career in research. However, I have gained knowledge and skills this summer that will carry over into any environmentally related career I pursue, and it is for this reason that I count my time in this position as a success.

- Nick Tippens

Trinity ’12

B.S. Earth and Ocean Sciences

Research is formalized curiosity. It is poking and prying with a purpose.

                                                              -Zora Neale Hurston

I don’t think I could have expressed as aptly my motivations for becoming involved in research through the Superfund Center as Zora Neale Hurston has just done. Even though the famous American anthropologist may have formulated this quote for reasons entirely unrelated to neurotoxicology research, I think it applies quite well in this context.

I have always been the girl with a lot of questions. When my Neuroscience professors lectured about certain theories, concepts and referred to various research studies, I always inadvertently inundated myself with waves upon waves of questions, stemming from curiosity, skepticism, plain confusion or a little bit of each. The problem was that I didn’t know what to do with all of it. My head almost became numb from the swirl and chaos of question marks incessantly pounding against my frontal cortex.

Research was the perfect solution. It channeled my hyperactive curiosity into the real world. It allowed me to find answers that I could never find in a textbook or in a lecture hall, at least not in the way I wanted to find them. The nicotine self-administration research I helped with during my sophomore year really opened my eyes to the promising state of drug development for smoking cessation treatment.  Having grown up watching my dad and uncle struggle to quit smoking, it was a relief to see that others like them may have an easier time of quitting in the near future. It made my relationship with science a little bit more intimate, and a little bit more meaningful. There was a definite purpose to it. During this summer, I have been actively involved in research that pertains to another issue that I feel very strongly towards: how commonly used or produced toxins in the environment can do irreparable damage (particular on a neurological level) to the environment, animals and most of all, to humans.

I have always been interested (or perhaps a better word is wary) about the long-term effects of exposure to toxins in humans. In particular, it is at once puzzling and alarming that many toxins, which are widely used and produced, can be so utterly underestimated and overlooked in its ability to be harmful to humans and animals alike. Perhaps we can attribute this ironic behavior to the fact that today, we live in a highly industrialized society, a fast-paced sort of world that has little patience for anything other than optimal efficiency and in turn, maximum output of product. One important example lies in agriculture. In order to increase agricultural productivity to support the rapidly growing world population, pesticides are conventionally used to rid crops of insects, thereby increasing harvest yield. While pesticides may be beneficial for many reasons, there are also caveats when it comes to using what are essentially poisonous substances, so frequently and ubiquitously.

How could we be so indifferent or unaware of inhaling and ingesting (as they are in our fruits, and vegetables) these toxic substances?! Well, this summer, before embarking on this Superfund internship in Dr. Levin’s lab, I had two general questions in mind: “At what levels of exposure to these pesticides do observable behavioral deficits appear?” and “Specifically what kinds of behavioral deficits are induced as a result of a determined threshold exposure of these toxins and how does it affect the overall functioning capacity of the organism?” In order to answer these questions (as well as many others!), I dived straight into the world of neurobehavioral teratology, in particular the study of how environmental chemicals and certain drugs of use can trigger brain-behavioral changes during embryonic and fetal development.

In Dr. Levin’s lab, we are specifically looking at two drugs: chlorpyrifos and dexamethasone. Chlorpyrifos is a commonly used organophosphate pesticide that is known to have negative effects on brain development. On the other hand, dexamethasone is a drug often given to babies born prematurely with lungs that have not fully developed. However, recent studies seem to suggest that dexamethasone can also cause developmental delay. Approximately 10-20% of people are exposed to dexamethasone.

Because both drugs have been linked to causing persisting impairments in cognitive and emotional responses, we decided to take a closer look at whether double exposure could intensify the severity of these neurological deficits. With the necessary controls in place, we subjected the rats to a series of behavioral assays. Through these behavioral assays, we can assess the cognitive functioning level of the exposed rats and compare the results to those of rats that have not been exposed to either drug or have only been exposed to one of the two drugs. This summer, I was primarily involved with running the rats in the radial arm maze.

For the maze, we would bait 12 of the 16 arms with fruit loops, and time how long it takes the rat to visit all of the baited arms. The radial arm tests both working memory (Mr. Whiskers: Have I already visited this arm?) and reference memory (Mr. Whiskers: Which arms are baited and which are not baited?). After getting the rats accustomed to this task, we began a series of drug-injection trials to determine the specific neural pathways that the rats are using when they are running the maze. Each drug targets certain receptors in the brain and blocks their function. Hence, if a certain drug really impaired their performance in the maze, we can be pretty confident that the neural pathway the drug is blocking has a role in the rats working and reference memory. So far, we are still in the process of analyzing the data derived from the radial arm tests, but whatever the results, I am sure that they will be very interesting and telling.

One important thing I have realized this summer as a Superfund research trainee, is that the research we are doing extends beyond the boundaries of the scientific field, and into the lives of ordinary people. It is a powerful tool for both environmental and social reform. Not only is research about poking around a maelstrom of hypotheses. It is also about “poking” legislators to adopt stricter regulations on these toxins, to incessantly motivate ourselves, as scientists, to devise techniques to clean up polluted sites or find safer ways to treat our crops. It is about “poking and prying” at the general public, to make certain that people know what their bodies are being exposed to: to inform. I think that is the most important thing of all.

If ever we had proof that our nation’s pollution laws aren’t working, it’s reading the list of industrial chemicals in the bodies of babies who have not yet lived outside the womb.

 

-Louise Slaughter, member of the US House of Representatives

Contrary to the name of the program, you don’t need to be an undergraduate student. This opportunity is open to undergraduate and Master’s students.

Visit this page to learn more about the research opportunities available and how to apply. The deadline for applications is March 15, 2013.

You can read about Dan and his research here.