FALL 2017

COVER STORY: From Clean Up To Cutting Edge Research


written by Sandra J. Ackerman | photos by Megan Mendenhall

The Atlantic KILLIFISH is not renowned for its size, cleverness, or appeal to the palate. Seafood restaurants don’t put it on the menu, and in sport fishing it’s considered bait, if anything. But in certain scientific circles, the killifish (Fundulus heteroclitus) is a celebrity because it offers new insights into evolution at work.

Large numbers of killifish live in the Elizabeth River, an estuary that runs through southeastern Virginia and empties into the James River. This has always been a busy waterway: the clearing of timber for shipbuilding, the transport of goods for trade, and naval battles from the Revolutionary and Civil wars have all left their marks on the area. But the biggest impact has come from the wood-treatment plants on the riverbanks that used creosote—an oily compound derived from coal tar—to protect valuable lumber from decay.

Rich DiGiulio

Over many decades, creosote passed into the water and settled in the riverbed, releasing large quantities of polycyclic aromatic hydrocarbons, or PAHs; these are compounds that have long raised concerns about public health because of their well-documented association with cancer. The Elizabeth River’s history of chronic contamination led the Environmental Protection Agency to designate it a Superfund cleanup site in 1990. To Richard Di Giulio, Sally Kleberg Professor of Environmental Toxicology, it also made the river a natural laboratory for his research.

When Di Giulio set out to find an animal model through which to focus his study of Elizabeth River contaminants, he didn’t have far to look. The Atlantic killifish was already a favorite of many researchers because of its hardiness and ability to adapt to a great variety of habitats. Best of all, individual killifish have relatively small home ranges and so are well-suited for studying the effects of contamination and other stressors at particular defined locations.

Di Giulio directs the Superfund Research Center (SRC) at Duke, so it might seem logical that he should do the fieldwork for his research at a Superfund cleanup site. However, this is only a coincidence; the two Superfund entities operate independently of each other.

Work done at different SRCs does not necessarily have to focus solely on contaminants at Superfund sites. Whereas the U.S. Environmental Protection Agency (EPA) oversees the cleanup of hazardous waste and reclamation of land at more than a thousand Superfund sites throughout the 50 states, there are relatively few Superfund Research Centers, and these are managed by the National Institute of Environmental Health Sciences.


The Duke University Superfund Research Center was awarded  a five-year grant for nearly $10.2 million from the National Institute of Environmental Health Sciences (NIEHS).

The grant will support five Superfund Center research projects investigating the later-life consequences of early-life exposures to hazardous chemicals. It will also fund six outreach and training programs designed to augment and support the center’s research.

NIEHS is part of the National Institutes of Health.

“This renewed funding will sup-port our center’s work to shed light on the long-term effects exposure to chemical pollutants can have on human and ecological health, and to develop approaches for reducing these exposures,” says center director Richard T. Di Giulio, Sally Kleberg Professor of Environmental Toxicology at the Nicholas School.

“It will also support efforts to share our research results with governmental, industrial and public stakeholders, engage communities in Superfund Center activities, and provide superior training for graduate students and postgraduate researchers,” Di Giulio says.

Highly interdisciplinary in nature, the Superfund Center brings together teams of biomedical, social and environmental scientists as well as engineers from across Duke’s campus—and from other institutions such as North Carolina State University—to investigate some of the most pressing issues in environmental health today.

Five-year funding for the SRC at Duke was renewed earlier this year (see story, page 6). It is one of only 18 SRCs currently in operation; each one has its own focus, says Heather Henry, administrator of the Superfund Research Program at the NIEHS. “A Superfund Research Center might be organized around a contaminant, such as arsenic or PCBs or PAHs, or around a mode of exposure, such as mining in arid regions,” she explains. “The Duke Center focuses on developmental exposure—an area that’s very much in need of additional research, because while an organism is still forming it can be very vulnerable to hazardous substances. In humans, it can take a long time to see the effects of early-life exposures, and the Duke Center has developed several innovative methods to help understand this phenomenon.”


The Duke Superfund Research Center is a highly productive training ground for young researchers from a wide variety of academic fields. Twenty-four former students or post- doctoral associates who received early- career training at Duke are now principal investigators at research labs and government agencies across the United States. This distinguished cohort includes 15 who are Duke alumni, including nine who received their degrees through the highly touted University Program in Environ- mental Health (UPEH), which celebrates its 35th anniversary this year.

AARON BARCHOWSKY PHD’85 University of Pittsburgh, Graduate School of Public Health

DANA DOLINOY PHD’07 University of Michigan, School of Public Health

EVAN GALLAGHER MEM’86, PHD’91 University of Washington, Department of Environmental and Occupational Health Sciences

CHRISTOPHER LAU BS’75, PHD’82 U.S. Environmental Protection Agency, National Health and Environmental Effect Laboratory

MAXWELL LEUNG PHD’12 California Environmental Protection Agency, Department of Pesticide Regulation

JENNIFER LYNCH PHD’04 National Institute of Standards and Technology, Chemical Sciences Division

JOEL MEYER PHD’03 Duke University, Nicholas School of the Environment

ALICIA TIMME-LARAGY PHD’07 University of Massachusetts Amherst, School of Public Health and Health Sciences

DAVID VOLZ PHD’06 University of California, Riverside, Department of Environmental Science


In the late 1980s the Elizabeth River had the dubious honor of being named the most polluted estuary in Virginia. Of all the industrial sites responsible, the Atlantic Wood treatment plant in Portsmouth, Va., was found to have contributed the most to the problem. Yet despite epic levels of contamination in their home waters, killifish that spend their whole lives at the Atlantic Wood site seem to maintain their abudance and ability to reproduce, although they do show high rates of liver cancer. When fish embryos from a relatively clean site nearby are exposed to polluted sediment, they develop deformed hearts, reduced circulation, and pericardial swelling, whereas embryos from Atlantic Wood parents show a near-complete resistance to these effects.

Di Giulio hypothesized the pollution itself was putting pressure on the killifish to change at a genetic level to adapt to their polluted environment—in other words, to evolve. In collaboration with a lab at Woods Hole Oceanographic Institution, he developed a DNA-based probe for investigating the role of specific genes in responses to toxicants and demonstrated that when the expression of particular genes is blocked, PAHs exert a greater or lesser toxic effect, depending on the gene.

As in humans, cancer in fish is a disease of old age and generally does not have a large effect on population dynamics. Di Giulio and his colleagues now think that the evolved resistance of Atlantic Wood killifish to PAHs involves genetic adaptations that reduce PAH effects during embryonic development of the cardiovascular system. Meanwhile, though, estimates of human risk from PAHs keep focusing on cancer—so are we overlooking another, equally important threat here?

A surprising discovery from the Elizabeth River killifish studies was the potent synergy between two of the most prevalent PAH types at the Atlantic Wood site. This synergy, the evolutionary driver of cancer resistance in the killifish population, is a crucial factor to take into account when we try to estimate levels of risk at other PAH-contaminated sites. From what we’ve seen with Atlantic Wood sediments, continuing to focus on the cancer-causing potential of PAHs could be dangerously misleading: we may end up with serious underestimates of other risks posed by some PAH mixtures.

Fortunately, federal, state, and town authorities, as well as local industries, have come together to begin limiting the release of pollutants into the Elizabeth River system and restoring the habitats to the extent possible. The Atlantic Wood site is now closed for remediation, while the nonprofit Elizabeth River Project (http://www. elizabethriver.org) is coordinating efforts to make the river swimmable and fishable by the year 2020 and to educate the public about the value of a healthy ecosystem.

Not far away in Chesapeake, research continues on the Republic Creosoting site, also “super contaminated,” according to PhD student Casey Lindberg. “One of the first things I did was assess the killifish at Republic Creosoting for their degree of resistance” to PAHs, she says. The fish here, like those at Atlantic Wood, showed few signs of heart deformities or elevated activity of the CYP1A enzyme; this population had developed a similar resistance to the health toll of creosote PAHs. But also like the Atlantic Wood killifish, the Republic Creosoting population seems to show that their evolved resistance comes at a cost: these fish could be extra-susceptible to other kinds of stress, such as a decreased amount of dissolved oxygen in the water.

These findings hold major implications—especially in the context of climate change, which brings drought to some areas and deluges to others. In estuaries like the Elizabeth River, abnormally high rainfall can rapidly lower the salinity of the water, which can be stressful for aquatic species. Rainfall also causes increased inputs of nutrients from land and agricultural runoff into the water, which, combined with increased temperatures, allows algae to bloom on the surface of the water. As the blooms start to die, the result is lower levels of dissolved oxygen for the fish that live underneath.

Five research projects will be supported by the new NIEHS grant. Each of these five projects will be augmented by the Superfund Center’s research support cores, research translation core, community engagement core and training core.

ONE “CHOLINERGIC AND MONOAMINERGIC MECHANISMS OF PERSISTENT NEUROBEHAVORIAL TOXICITY” which is aimed at identifying how exposure to diazinon, triphenyl phosphate and other neurotoxins affects an organism’s neurotransmitter system and can result
in persisting cognitive and emotional dysfunction.

which is aimed at understanding how the regulation of key receptor pathways in the body may help mediate later-life skeletal malformations, obesity and other harmful effects of early exposure to halogenated phenolic compounds (HPCs), chemicals which mimic hormones on the body.

THREE “PERSISTENT MITOCHONDRIAL AND EPIGENETIC EFFECTS OF EARLY-LIFE TOXICANT EXPOSURE” which is aimed at identifying whether the toxic effects of certain chemicals on mito-chondrial function are highly persistent or inheritable; and if these effects are greater among certain genetic backgrounds.

which is aimed at exploring how low-level exposures to polycyclic aromatic hydrocar-bons (PAHs) affects physical, behavioral and metabolic development, and how PAHs have driven evolution in free-living populations of fish.

which is aimed at devising new biological techniques – using an understudied group of micro-organism known as non-basidio-mycete fungi – to remediate high levels of PAHs and other developmental toxicants that can accumulate in sediment.


If some animals—say, those with short generation times—can sometimes evolve to thrive in the face of chronic pollution, does this mean the pollution is benign? Probably not. In several subpopulations of Elizabeth River killifish, the genetic structure has been permanently altered, and there’s evidence that the evolutionary adaptations that let them live with the pollution have come at a cost to their overall fitness. Di Giulio explains, “We are currently performing studies exploring fitness costs—i.e., showing that the adapted populations are more sensitive to elevated temperature and hypoxia. These studies involve detailed analyses on mitochondrial function, aerobic performance and behavior.”

Joel Meyer

Joel Meyer, who directs the SRC’s training core, carries out studies like this at the level of genes or molecules. “I’m looking at how environmental agents can cause damage to DNA and at the molecular processes that organisms employ to prevent and/or repair DNA damage,” says Meyer, Truman and Nellie Semans/Alex Brown & Sons Associate Professor of Molecular Environmental Toxicology. He also looks for the subtle genetic differences within a species—or human population—that may make certain individuals more susceptible, or else more resistant, to DNA damage.

In many cases he finds such differences among genes that affect an animal’s mitochondria, self-enclosed bodies within each cell that produce and store energy. Mitochondria are unique in having their own genome, which is far smaller than the animal’s overall genome and lacks some of its pathways for repairing DNA damage. For this reason, toxicity and diseases that arise from mitochondrial damage can be very serious.

Says Meyer, “Along with a lot of other labs, we have observed that if you have a stressor that affects mitochondrial function, the damage may become apparent only over the long term, and sometimes even across successive generations. We’re now setting out to test the possibility that certain toxins in the environment can cause these types of long-term damage, and to find out whether genetic differences may make some individuals more susceptible than others.”

Jamie Harris, a Duke undergraduate in the class of 2020, is enjoying her summer job in Meyer’s toxicology lab. “This is really different from the work I did in an aging lab while I was in high school,” she says. One of her experiments calls for observing microscopic worms (C. elegans) under a plate reader after they have been exposed to extracts of sediment from the Elizabeth River, to look for possible mitochondrial damage. Harris enjoys the ingenious design of this assay: “We use a certain strain of C. elegans that contains a fluorescent protein in its genome, so the worms glow when you put them under the microscope and in the plate reader,” she explains. “It’s really cool: these worms are too small to see with the naked eye, but they can still tell us a lot about how toxins affect mitochondrial function.”


The deputy director of Duke’s SRC, Heather Stapleton, studies the environmental health profile of quite a different habitat—the typical American home, with its vast assortment of furnishings, appliances, clothing, tools, and devices, along with varying amounts of grime and dust. Earlier this year Stapleton and her coauthors briefly became world-famous for their chemical analysis of household dust, in which they found surprisingly high levels of endocrine-disrupting compounds (EDCs). Stapleton is Dan and Bunny Gabel Associate Professor of Environmental Ethics and Sustainable Environmental Management.

Heather Stapleton

Many of the newspaper headlines oversimplified the research to the point of silliness (“Is household dust making you fat?”), but the health effects of EDCs should be taken seriously, Stapleton says. The compounds she and her colleagues identified are known to interfere with the human hormonal system that prompts the formation of both bone cells and fat cells; thus, as with many other substances being studied at the SRC, exposure to them early in life poses the greatest risk.

Stapleton’s main line of investigation focuses on the flame-retardant substances commonly used today to treat clothing, rugs, mattresses, and many other household objects. All too often these substances include polybrominated diphenyl ethers (PBDEs)—compounds that can interfere with the thyroid system, which normally regulates human metabolism, growth, temperature control, and a host of other important functions. Exposure to PBDEs also appears to raise women’s risk of developing papillary thyroid cancer.

Of course it’s a good idea to minimize the risk that a child’s pajamas will burst into flames if he gets too close to the toaster, but Stapleton thinks this goal can be met without the addition of other health hazards. Some new and improved flame-retardant substances are in the works, but she would like to see a more basic response to this public-health threat. “The first thing we ought to do is determine exactly where we need flame retardants and where we don’t,” she says.


Professor Ed Levin holds appointments at Duke’s medical school and undergraduate college as well as at the Nicholas School. Within the Superfund Research Center, at his neurotoxicology lab, he’s trying to understand how exposure to harmful substances may affect the brain, not just in terms of one chemical signal but among several of them: dopamine, serotonin, and acetylcholine. Sorting out these interactions is a challenge, he admits, but says, “Even at that, it’s just three neurotransmitters among many others.”

Levin’s lab works with a range of model systems, from in-vitro cultures to invertebrates to aquatic organisms (zebrafish) to mammals (rodents). The different models complement one another, he says.” In zebrafish, for example, his research team is studying how exposure to certain toxins early in life can lead to adverse effects later on, whereas in rodents they are looking for possible new treatments to alleviate such effects.

Andrew Hawkey, a postdoctoral associate in Levin’s lab, studies the neurotoxic effects of the pesticide diazinon, which was once widely used in the United States but is now applied only in agriculture. “Our interest is in off-target effects, because the insects targeted by diazinon are just the first organisms exposed to it,” he explains. Farmers and agricultural workers, people who live near areas where diazinon is used, and consumers of the food that’s grown with it all run some risk of being affected by this compound.

Diazinon is designed to work in insect nervous systems, but Hawkey wants to find out how it affects it the brains of mammals at their most vulnerable stage: before birth, while they are still developing. He implants an osmotic mini-pump under the skin of pregnant female rats to administer a consistent, low level of exposure throughout pregnancy. (A “control” group of pregnant rats is fitted with pumps containing an inert substance.)

“In the offspring of these rats, we’ll be looking for evidence of anxiety, changes in behavior or cognition, and changes in activity,” Hawkey says. Meanwhile, two partner labs at the SRC are studying the neurochemistry, looking closely for changes in various signal receptors in the brain that may correlate with changes in behavior or cognition.

Hawkey’s previous research had focused on addiction: how alcoholism or substance abuse affects the user and, if she’s pregnant at the time, how it ultimately affects the child. Coming to Duke, he says, gave him a different perspective. “Now I’m looking at how chemicals that people are exposed to inadvertently can affect them and their offspring,” he says, adding, “It’s been really fascinating to me, looking at questions that wouldn’t have been posed in this fashion in my old field. It has definitely expanded my perspective on the brain.”


Another major research project at the Duke SRC, led by Claudia Gunsch in the Pratt School of Engineering, focuses on developing innovative ways to clean up, or remediate, polluted environments. Each instance of pollution has its own ecology and industrial history, as well as current and future challenges, so each solution must likewise be customized for the problem at hand. This approach calls for scientific creative scientific thinking. One striking example combines tools from physical and chemical engineering, microbiology, and chemistry, using a little-known group of micro-organisms to lower the harmful levels of PAHs and other developmental toxicants that can accumulate in the sediment of polluted waterways.

The intensive training for future scientists and the multidisciplinary approach of most research projects are two of the elements that makes the Duke Superfund Research Center a hub of enriching experiences. A third element is the SRC’s strong program of community outreach, which includes public talks, meetings with residents in pollution-affected areas, and social media campaigns.

Lindsay Holsen, an undergraduate from Lawrence University, recently finished a summer internship at the Duke SRC, dividing her time between research in Joel Meyer’s lab and community-outreach projects. In the latter area, her Spanish-language skills were an asset. “I helped translate some of the Center’s material on contaminants in fish and in garden soils with the help of two native Spanish-speakers,” she says.

Holsen feels she learned a lot about the challenges of communicating complex research and about the need for balancing costs with solutions. “There are a lot of temporary solutions to environmental problems, but you have to think about solutions for the long term and ensure that the affected community is involved in the process,” she says.

Encouraging this kind of thoughtful approach to environmental-health challenges is an investment in the future that NIEHS administrator Henry particularly appreciates. As she sees it, “The fact that the Duke SRC is an integrated program, with health, technology, and community engagement, makes it a unique experience for trainees, allowing them to get a real depth of knowledge. While their program is grounded in specific [fields of] expertise, it also helps them understand how their work fits into the bigger picture.”

Sandra J. Ackerman is a science writer based in Durham, N.C., and is a member of the National Association of Science Writers.