Expanding the vision of environmental health at UNC-CH.

In a town that devotees call the "Southern Part of Heaven," the University of North Carolina at Chapel Hill (UNC-CH) is known nationally for its schools of public health, medicine, and pharmacy. These schools are physically just across the street from one another, but they can seem worlds away if their knowledge and resources aren't shared. To bring these researchers together, the Center for Environmental Health and Susceptibility (CEHS) was funded with a $3.78 million, four-year grant from the NIEHS to become one of 22 Environmental Health Sciences Centers. "We have an unusual mix of basic, clinical, and population scientists," says CEHS director James Swenberg, a UNCCH professor of environmental sciences and engineering. "The center's goal is to, by bringing these groups together, expand our vision of environmental health research."

Center researchers work to understand the fundamental processes that contribute to chemical toxicity and to combine this knowledge with epidemiology to reduce environmental disease. Increasingly, work at the center focuses on understanding how environmental and genetic determinants of disease work together in populations. And whereas researchers have in the past focused largely on average susceptibility when looking at the distribution of disease among a particular population, CEHS scientists are among those now looking at populations with greater- or lesser-than-average disease susceptibility, Swenberg says. Furthermore, they are trying to help people understand what role their genetic makeup plays in susceptibility.

That expanded vision can be seen in the center's recent. growth, in April 2004, from three research cores to five. The center also includes four facility cores to offer services such as high-throughput genetic analysis, provision of a variety of biomarkers, and expertise on statistical design and analysis. And the Community Outreach and Education Program (COEP) brings what researchers are learning to the people of North Carolina and beyond.

Capitalizing on an Investment in Genomics

The genomics revolution has contributed to the fast growth of the CEHS. In the past few years, UNC-CH has made a large investment in genome science, including creating a Department of Genetics in its School of Medicine and building a proteomics facility. Partly because of the ability to examine a greater number of samples faster than ever before, researchers are no longer looking at single-nucleotide polymorphisms in isolation. "We're learning that it's much more complex than that," Swenberg says. "Now researchers are moving toward looking at haplotypes--how groups of genes are interacting to give greater or lesser susceptibility." One of the CEHS's new research cores, the Transomics Research Core, directed by genetics researcher David Threadgill, works to capitalize on these investments by applying new technologies such as genomics, proteomics, and metabolomics to the understanding of environmental health risks.

Recent work by geneticist Charles Perou is an example of this trend. Using microarrays, which produce fluorescent images that allow researchers to monitor the expression of 20,000 genes at once, Perou is able to look for patterns of gene expression in thousands of tumors. He has shown that human breast tumors arise from at least two distinct types of cells--basal and luminal epithelial cells--and that basal cell carcinomas are less responsive to chemotherapy.

Perou has found that the basal subtype does not respond as well to p53, the "guardian" protein that induces programmed cell death in response to DNA damage. This difference may partly explain the basal subtype's resistance to treatment such as chemotherapy, which involves induction of apoptosis. Perou is also working with other investigators in the Toxicogenomics Research Consortium, funded by the NIEHS, to use microarrays to study patterns of cellular response to environmental toxicants such as those in car exhaust and cigarette smoke.

Closely related to the work of Transomics Research Core is that of the Genetic Susceptibility Research Core, directed by William Kaufmann, a professor of pathology and laboratory medicine. This core seeks to find out how variations in DNA from individual to individual affect the risk of disease caused by environmental exposure. The core's work focuses on how gene-environment interactions affect DNA repair and genetic susceptibility to diseases such as cancer, asthma, and heart disease.

Using Chinese hamster cell lines, environmental scientist Jun Nakamura, collaborating with Yoshiko Kubota of Japan's Akita Medical University, found that only one out of several identified polymorphisms in XRCC1--a gene suspected of playing a role in breast cancer--had a functional effect. Nakamura, Kaufmann, Swenberg, and epidemiologist Bob Millikan applied this finding to their work with human cell samples from the Carolina Breast Cancer study, which is led by Millikan. Early findings suggest that this polymorphism in XRCC1 increases the risk of breast cancer. Swenberg stresses that these are preliminary results, but that "it's an example of how bringing together epidemiologists, basic researchers, and clinicians can lead to new discoveries."

Exposure in the Most Vulnerable

Researchers in the Developmental Susceptibility Research Core examine the effects of exposures during the most vulnerable periods of human development: studies are investigating how environmental exposures from preconception through childhood contribute to such effects as miscarriage, birth defects, developmental deficits such as autism, and childhood diseases such as cancer. "The collaborations facilitated by the center make possible many new projects," says core director Andrew Olshan, a professor of epidemiology.

Core accomplishments include findings from nutritionist Steven Zeisel, published in the 12 March 1999 issue of Developmental Brain Research, that show that inadequate choline (a nutrient found in milk and eggs) in the diet during pregnancy alters fetal brain biochemistry, with resulting deficits in memory and mental processes that last throughout the offspring's lifetime. In the March-April 2001 issue of Developmental Neuroscience, Zeisel and colleagues described how choline supplementation during pregnancy influences development of the hippocampus--which governs learning and memory, among other functions--by altering the timing of the creation and differentiation of progenitor cells known to be associated with these functions in the adult brain. In unpublished research, Zeisel has also shown that diethanolamine (a chemical used in shampoos, lotions, creams, and other cosmetics) can induce choline deficiency in mice. He is doing further research to determine whether frequent use of this compound causes abnormal brain development in offspring of exposed mice.

In other core work, Stephanie Mulherin Engel, now a postdoctoral fellow at the Mount Sinai School of Medicine, studied the role that inflammatory cytokines (messenger chemicals produced inside the body in response to infections and toxicants) play in preterm and small-for-gestational-age births. For her doctoral dissertation, Engel worked under the direction of Olshan and collaborated with David Savitz of UNC-CH and Stephen Chanock, an investigator at the National Cancer Institute, to confirm that some known combinations of polymorphisms in cytokines increase the risk of spontaneous preterm birth; the team also identified others that had not been previously studied.

"Preterm birth is one of the most important contributors to infant mortality, but the causes are unknown," Olshan says. "This work gives us more information about the inflammation pathway that is suspected to affect preterm birth and about the genetic variants that may, in combination with exposures, affect a woman's risk of preterm birth." Savitz, Mulherin, and Olshan, along with obstetrics and gynecology professor John Thorp, recently submitted an NIH grant proposal to further examine the impact of exposures, genetic variation, and other factors on cytokine levels in pregnancy, especially among African Americans.

Investigators in the Toxicokinetic Susceptibility Research Core study the mechanisms of how toxicants found in air, water, and food are processed by the body, and how toxicants cause greater effects in some people than in others. Stephen Rappaport, a professor of environmental science and engineering, directs this core.

One high-profile example of this core's work is the ongoing Long Island Breast Cancer Study Project, one of the largest environmental epidemiological studies conducted on breast cancer. Led by CEHS deputy director Marilie Gammon, researchers examined 1,000 blood samples from newly diagnosed breast cancer patients and controls. The study focused on biomarkers of exposure to polycyclic aromatic hydrocarbons (PAHs), which are by-products of combustion found in air pollution, cigarette smoke, cooked foods, and other sources. These markers, called PAH-DNA adducts, are alterations to DNA that are considered precursor lesions to cancer. One report from this study, published in the August 2002 issue of Cancer Epidemiology Biomarkers & Prevention, showed that women with the highest quartile of PAH-DNA adducts had a 50% increased risk of breast cancer.

But the study did not find a simple cause-effect relationship. Subjects with the highest levels of exposure to PAHs did not necessarily have the most PAH-DNA adducts or the highest risk of breast cancer. Gammon says, "Our data indicate that PAH-DNA adduct formation may influence breast cancer development, although the association does not appear to be dose-dependent and may have a threshold effect. The risk for breast cancer in relation to PAH-DNA adducts did not vary when we examined whether someone was a smoker or nonsmoker, or when the subject reported eating grilled and smoked foods or not. So you can take two people with the same exposure levels, and one gets adducts, the other doesn't." Recent findings based on data from more than 1,800 women confirm these results; this report will appear in an upcoming issue of Archives of Environmental Health. The team is continuing to study these issues, focusing on the idea that individuals have different responses to environmental exposures because of their genetic makeup or other factors.

The center's second new research effort, the Obesity Research Core, directed by associate professor of nutrition and medicine Joyce Harp, includes researchers from nutrition, medicine, pediatrics, epidemiology, surgery, and other fields who will study how the environment impacts the development and maintenance of obesity. For instance, many compounds such as the pesticide DDT are lipophilic (easily dissolved and stored in fat). When someone who is obese loses a large amount of fat, such as in the increasingly common gastric-bypass surgery, what happens to the lipophilic compounds?

"Do these chemicals just redistribute into the remaining fat? That is what we hypothesize," Swenberg says. "Do the levels of environmental toxicants triple or quadruple if someone loses [hundreds of pounds]? We don't know. We'd like to find out something about the toxicokinetics, ... exactly what happens." In addition to Swenberg, other core members involved in these studies include Zeisel, lipid biochemist Rosalind Coleman, and surgeon Timothy Farrell.

Other researchers will study obesity-related alterations to the immune system that may affect a person's response to infection, cancer susceptibility, and other characteristics. Researchers also are investigating the environmental factors that contribute to obesity, including nutrition and the built environment--the density and proximity of recreation facilities, transportation options, and pedestrian walkways.

Dispelling Genetic Myths

Drawing on the center's extensive research in breast cancer and other conditions, staff of the COEP, directed by environmental sciences and engineering professor Frances Lynn, have conducted workshops and other outreach programs throughout North Carolina. "Center scientists are intimately involved in all the programs we design for the public," Lynn says. Many of the programs help people better understand how their genes and their environment interact to affect their disease risk. For instance, Lynn obtained a supplemental grant from the NIEHS to develop materials on gene-environment interactions and ethical dilemmas posed by genetic testing (such as employment discrimination based on a person's genetic data and the psychological impacts of genetic testing).

Through conducting workshops across the state, Lynn found that many women believe that inherited mutations in genes play a large role in explaining the occurrence of breast cancer. Lynn and her staff draw on CEHS research by Gammon, Millikan, and others to dispel that myth. "Only five to ten percent of breast cancers are caused by a woman's inherited genetics, and an even smaller percentage is thought to be explained by BRCA1 and BRCA2, the so-called breast cancer genes," Lynn says.

COEP staff tailor programs to various audiences. Senior citizen groups have responded well to "Reduce Breast Cancer Bingo," in which players fill their cards by answering questions about the role that various factors such as obesity, alcohol consumption, and exposure to PAHs in air pollution and environmental cigarette smoke play in breast cancer risk. With other groups who are interested in more scientific detail, participants read and discuss case studies of three fictitious women who show various risk factors for breast cancer and the ethical dilemmas that genetic testing can raise.

In addition, the COEP has developed a middle- and high-school science curriculum, "Our Genes, Our Environment, Our Health," and conducted workshops with 78 science teachers and 85 students in North Carolina. The curriculum complies with the North Carolina Standard Course of Study and includes activities in which students focus on ethical issues.

COEP science educator Michele Kloda stresses the importance of sharing the center's research with students and teachers: "North Carolina teachers are required to teach students the science behind gene-environment interactions, but very often the information they have access to is out of date or unrelated to students' lives. In contrast, we can provide teachers with current center research and activities about heart disease, tobacco smoke exposures, and obesity, and ultimately make environmental health science more relevant and accessible to young adults."

Investing in the Future

The center places great emphasis on fostering young investigators, providing junior investigators with formal grant-writing instruction and targeting its pilot project funding program toward them. Swenberg hopes this emphasis will mean more future growth for the center and for environmental health science as a whole. "We feel that bringing junior investigators into environmental health research is a very critical responsibility of the center," he says.

Those efforts do seem to be paying off. In fiscal years 2001 and 2002, recipients used $165,000 in CEHS pilot funds to garner a 22-fold return of more than $3.6 million in external awards. CEHS members are directing eight nationally competed training grants, three of which are funded by the NIEHS.

Lilian Calderon-Garciduenas, a recent Ph.D. recipient in toxicology and an advanced postdoctoral fellow in Swenberg's lab, is a recent example of these successes. She used a CEHS pilot project grant to begin research that suggests that severe air pollution may contribute to neurodegenerative disease. In the May 2002 issue of Toxicologic Pathology, Calderon-Garciduefias and colleagues report on their study of dogs in Mexico City, which is known for its severe air pollution. Their results indicate that otherwise-healthy lifelong canine residents show evidence of changes in the frontal cortex, hippocampus, and olfactory bulb, compared to dogs from a nonpolluted rural area. These changes include upregulation of some inflammatory cytokines and genes such as COX-2, which is related to inflammation, and increases in amyloid-beta, which is a precursor lesion of Alzheimer disease.

Calderon-Garciduenas will study this further by looking for similar changes in human brains and by investigating how the changes occur. In collaboration with CEHS epidemiologists, she will use geographic information systems to look at the distribution of cases to find out what types of pollution may be more likely to cause neurodegenerative damage. In a separate unpublished study, Calderon-Garciduenas and colleagues found that air pollution may be related to chronic pulmonary hypertension; among 48 children who resided in Mexico City, 29% showed evidence of high pulmonary pressure. The epidemiological impact of this finding in a city where 6 million children live needs to be addressed, says Calderon-Garciduenas.

"These findings have huge implications for environmental health," Swenberg says. "And this entire research program was funded out of a pilot project from our center. We think that encouraging young people to go into environmental health research and helping them have success during their early years of research is very likely to make them lifelong environmental health researchers.