United States
       Environmental Protection
       Research and Development

       National Health and
       Environmental Effects Research

       Science Report

       Volurne 2, Issue 1
       March 2002


Click on title to link directly to article.


New Cell Line Detects Endocrine Activity	3

Competition for Resources Influences Plant Diversity	4

Bromoacetic Acid Affects Male Reproduction and Fertility in Rats	5

Heptachlor Exposure Causes Subtle, Persistent Deficits in Rats	6

Relationship of Dioxin Exposure to Sanitary Products and Endometriosis Studied	7

Study Launched on How Microorganisms Cause Gastroenteritis	9

Postdoc Co-Authors Three Papers on Toxicoqenomics	10


EMAP Technology Transferred at Coastal Assessment Workshop	11


EMAP Symposium 2002 Coming May 7 - 9	12


Atlas Provides Scientific Basis for Land Use Planning	13

Dioxin Toxicity Equivalence Factors in Fish and Wildlife Published	14


New Cell Line Detects Endocrine Activity

       A new cell line has been developed by the National Health and Environmental
Effects Research Laboratory (NHEERL) that may be useful in rapidly screening large
numbers of chemicals to identify those with endocrine-disrupting properties. In recent
years there has been a growing concern about the increasing number of chemicals
shown to disrupt endocrine activity affecting the reproductive systems of humans or
wildlife.  Protocols are being developed by the Reproductive Toxicology Division  for the
screening of chemicals that are potentially endocrine-active. Led by Dr. Vickie Wilson
and Kathy Bobseine, research to develop the new cell line is published in the March
2002 issue of Toxicological Sciences.

      A breast cancer cell line, MDA-MB-453, was used as a starting point. These
immortal cells contain binding sites for androgenic (male) hormones.  When the cells
were treated to alter their DNA, they were transformed into a new cell line called
MDA-kb2.  The change in  DNA remained stable over many generations of cells grown
from this line. Subsequent exposure of the new cell line to chemicals with male
hormone-like properties induces a change at the binding sites that can be measured
rapidly and quantitatively.

      The MDA-kb2 cell line will be  made available to the American Type Culture
Collection, which guarantees safekeeping and distributes cell line samples to
researchers worldwide at a nominal charge.  This cell line has the potential to be used
to screen large numbers of chemicals and to prioritize the use of more expensive and
time-consuming in vivo assays using animals.

Competition for Resources Influences Plant Diversity

      A clearer view of how plants compete for resources and how this competition
influences the species composition of a community is provided in a paper published in
the Jan. 3, 2002, issue of the journal Nature by Dr. Robert McKane of the Western
Ecology Division (WED), the lead author of 11 investigators. The paper documents, for
the first time, that species diversity and dominance in a plant community is closely
linked to how the plants divide a single limiting below-ground resource-nitrogen, in this

       The field work and laboratory analyses were funded by a grant from the National
Science Foundation when McKane was employed at the Marine Biological Laboratory in
Woods Hole, Massachusetts, but he did the data  analyses and wrote the paper after
joining WED in Corvallis, Oregon.

      At Toolik Lake, near Alaska's Arctic National Wildlife Refuge, the researchers
studied tussock tundra plants whose growth is limited primarily by the availability of
nitrogen in the soil.  They found that the plants in  this community took up nitrogen at
different times, depths, and in different forms.  They also found that the most productive
plant species, cottongrass, used the most abundant forms of nitrogen, while less
productive species used less abundant forms. Cottongrass is primary forage for caribou
and other arctic wildlife, but has declined in abundance in recent decades. The decline
coincides with recent increases in arctic temperatures that may alter the availability of
different forms of soil nitrogen.

      Ecologists have long been interested in how species living in the same
community divide limited resources and thereby promote diversity by reducing
competition.  This resource partitioning, or niche differentiation, is known to be an
important determinant of species diversity and composition in animal communities.
However, its importance in structuring plant communities has been difficult to determine,

primarily because of difficulties in measuring how plants compete for below-ground

      The researchers overcame that problem by performing more than 15,000
injections of tundra plots with 15N, a stable isotope of nitrogen, and analyzing more than
2,000 plant samples for isotope uptake.  By injecting different chemical forms of 15N at
different times and soil depths, they discovered how plants took up naturally occurring
forms of soil nitrogen.  The study provides a clearer view of below-ground competitive
interactions than existed previously, and establishes a means to better address
fundamental questions about plant species diversity.

Bromoacetic Acid Affects Male Reproduction and Fertility in Rats

      The haloacid, bromoacetic acid (BCA), one of the more prevalent disinfection by-
products (DBPs) that can form when chlorine is used in water to kill pathogens, has
been found to cause adverse effects on sperm production in rats. The Reproductive
Toxicology Division (RTD) studied the noncancer effects of BCA on reproduction in
male rats and compared the results to earlier work on another prevalent DBP,
dibromoacetic acid (DBA), which showed that treating adult male rats produced adverse
effects on sperm production.

      Scientists reported that BCA, like  DBA, also adversely affected the formation of
normal sperm and the fertility of treated male rats. Fertility was decreased about 25-30
percent at all BCA doses used, even the lowest one. These results are published in the
January/February 2002 issue of the Journal of Andrology, where they are also featured
in photographs on the cover.

       The fertility of treated rats was also compared to the level of a specific sperm
protein, SP22, found on the surface of sperm. A decrease in the level of SP22 was
present in all dosage groups and was correlated with a decrease in fertility in treated

rats. The study confirmed that SP22, a protein identified earlier by RTD, is a useful
biomarker for fertility, although its application in assessing human infertility is as yet

       These results suggest that reproductive alterations in male rats are likely even at
much lower dosage levels. Therefore it is important to determine additivity effects
associated with exposure to low-dose mixtures of haloacids, a condition that better
represents how humans are exposed. Future studies at RTD will assess whether
effects on SP22 and fertility of rats are additive when low-dose mixtures of haloacids
are used. Under a Cooperative Agreement with the University of North Carolina at
Chapel Hill, an epidemiology study is looking at the effects on the fertility of sperm from
men drinking water with varying levels of  DBFs; SP22 will be one of the sperm
parameters measured.  Other Cooperative Agreements with Research Triangle Institute
and Colorado State University are studying fertility and SP22 levels in male offspring of
mother rats exposed during pregnancy to low doses of BCA and DBA, respectively.
The results of all of these studies will contribute to decisions on whether the Maximum
Contaminant Level for haloacids is protective enough for human  exposure.

Heptachlor Exposure Causes Subtle, Persistent Deficits in Rats

      Two  recent publications by scientists in the  Neuortoxicology Division (NTD)
revealed subtle but long-lasting behavioral and neurological effects of the cyclodiene
pesticide heptachlor on  rat development.  Although heptachlor was banned in the
1980s, it is still found in food  samples and is considered a "persistent organic pollutant"
by the United Nations Environment Programme. These results are particularly relevant
because other cyclodiene pesticides are in restricted commercial use today.  The
research was partially supported by the Hawaiian Heptachlor Research and Education
Foundation from a trust fund  established after routine monitoring discovered high
exposure to heptachlor from contaminated cow's milk on the island of Oahu in the

       Using doses similar to levels found in human breast milk samples taken in 1981
in Oahu, rats were exposed to heptachlor before birth and for several weeks after birth.
As described in the April 2001  issue of Toxicological Sciences,  NTD researchers found
a delay in behavioral development in young  rats and, later, an impairment of a learning
and memory task when those rats reached adulthood.

      Also investigated was the possibility of increased susceptibility to
neurodegenerative diseases after developmental exposure to heptachlor. The doses
used were no more than 10-fold greater than those in the behavioral study, and were
used over a shorter dosing period.  As published in the December 2001 issue of
Toxicological Sciences, rats treated with  heptachlor during early stages of development
showed neurochemical changes as adults.  They had increased binding to the
dopamine transporter (DAT) receptor sites, implying an increase in receptor levels.  The
DAT allows entry of other neurotoxicants into neurons, and may also be involved in
maintaining neuronal integrity. Heptachlor exposure during neuronal development
increased DAT binding during early development, adolescence, and adulthood. Similar
results were obtained with dieldrin, another cyclodiene pesticide.

      It is hypothesized that these neurochemical changes may increase susceptibility
to other neurotoxicants, which may cause degenerative diseases, such as Parkinson's
disease, later in life. Further research is  critical to understanding the potential
influences of developmental toxicant exposure on neurological and degenerative

Relationship of Dioxin Exposure to Sanitary Products and Endometriosis Studied

      Two papers by NHEERL scientists in the January 2002 issue of Environmental
Health Perspectives address whether sanitary products are a source of dioxin and
whether endometriosis is associated with dioxin exposure.  Dioxins are a family of long-
lasting, bioaccumulating, toxic chemicals found in the environment largely as a result of


manufacturing and combustion processes. This family, which includes some
polychlorinated dibenzodioxins, dibenzofurans, and biphenyls, is known to affect human
and animal health in many ways, including effects on the endocrine and immune
systems. The most toxic member, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD or
"dioxin"), is frequently used as a reference compound in toxicity studies.

      There is particular concern about possible effects on infants and children from
exposure to dioxins in diapers.  The media also have raised concern about possible
links between dioxin exposure from tampons causing endometriosis, a disease of the
reproductive system that affects millions of women in the United States.

      A study published by Dr. Michael DeVito of the Experimental Toxicology Division,
in collaboration with  Dr Arnold Schecter of the Environmental Sciences Discipline,
University of Texas School of Public Health at Dallas, concluded that although
exceedingly low concentrations of dioxins were found in the diapers and tampons
sampled, these sanitary products made in the United States do not contribute
significantly to dioxin exposure.  For comparison, the estimated dietary exposure of
infants to dioxins is approximately 30,000 to 2,200,000 times greater than their
exposure from diapers.  The estimated dietary exposure of women to dioxins is
approximately 13,000 to 240,000 times greater than from tampons.

      In the second paper Dr. Linda Birnbaum of the Experimental  Toxicology Division
and Dr. Audrey Cummings of the Reproductive Toxicology Division critically reviewed
the literature of the past nine years on the relationship between exposure to dioxins and
an increased incidence of endometriosis,  and they suggest a possible connection.  But
the hypothesis that dioxins are causally related to endometriosis needs further study.

      Studies on monkeys and rodents have linked exposure to TCDD and other
dioxins to an increased incidence or promotion of endometriotic lesions. Recent
epidemiological studies also suggest a link between these  chemicals and the incidence

of the disease in humans, but these conclusions remain controversial because there is
insufficient relevant information in the literature on which to base a definitive judgment.

Study Launched on How Microorganisms Cause Gastroenteritis

      A study on gastrointestinal disease in Texas  is part of a large research effort by
the Human Studies Division to determine the extent and causes of waterborne disease
in the United States.  On average, adults in the United States have one to two episodes
a year of gastroenteritis (diarrhea and vomiting), but young children and elderly adults
have six to 10. Although most cases are caused by any of a wide variety of
microorganisms, how people come in contact with these disease-causing agents is
rarely known. The Southwest Texas Enteric Epidemiology Study, sponsored by EPA
and starting in March 2002, will attempt to determine how microorganisms are
transmitted to humans and cause gastrointestinal disease.  Dr. Rebecca Calderon of
the Human Studies Division is the EPA Project Officer.

      The Texas study will determine the incidence of gastrointestinal illnesses in two
areas that get their drinking water from different sources. Participants in the Eagle Pass
area use Rio Grande water for drinking, whereas participants in the Del Rio area use
spring water.  Up to 150 families in each of the two communities will be studied. The
families will record their own episodes of gastroenteritis, as well as contacts with
infected persons, food, drinking water, recreational water (such as swimming and
wading pools), and animals, all  potential risk factors for gastroenteritis. From these data
the researchers will be able to estimate how much gastrointestinal illness is associated
with each potential source of infection.

      Data collection will be completed by the summer of 2003, and preliminary results
are expected by December 2003. Field work on similar studies in Massachusetts and
Washington has been completed, and one in Iowa will conclude this spring. Preliminary
results are expected by the end of 2002. Data from these studies will be used to make

national estimates of waterborne disease, as mandated by the Safe Drinking Water Act
Amendments of 1996.

Postdoc Co-Authors Three Papers on Toxicogenomics

      Understanding the development of cancer cells in humans and animals is the
focus of three papers on toxicogenomics. Extending work done under the mentorship of
Dr. Anthony DeAngelo of the Environmental Carcinogenesis Division (ECD), Dr. Lynn
Crosby co-authored three articles on toxicogenomics recently accepted for publication.
She is an EPA Postdoctoral Fellow in ECD from the Curriculum in Toxicology at the
University of North Carolina at Chapel Hill. Other authors are from the National Institutes
of Environmental Health Sciences,  Duke University Medical School, and
GlaxoSmithKline Inc., all in North Carolina's Research Triangle.

      The emerging field of toxicogenomics studies the response of cells to stress at the
most basic level: the molecules within the cell. The cell may respond by adaptations
such as DNA repair with accompanying temporary growth cessation; the production of
molecules that counteract the stimulus, such as cytokines that fight infection; or cell

      One  paper, to be published in the July/August 2002 issue of Toxicologic
Pathology, describes the development of a 7-gene oxidative stress test on cultures of
human liver cancer cells.  Because oxidative stress is a consequence, and  possibly also
a cause, of many adverse health outcomes, including Alzheimer's, Parkinson's disease,
and cancer, this relatively quick, efficient, and cost-effective screen to detect the
oxidative stress potential of various substances will benefit many  stakeholders.

      Related review articles,  published in the January/February 2002 issue of
Toxicologic Pathology and to be published in a spring 2002 issue of Human and

Ecological Risk Assessment, address the role of toxicogenomics in pathology and drug
discovery and in risk assessment, respectively.

                          TECHNICAL ASSISTANCE

EMAP Technology Transferred at Coastal Assessment Workshop

      As part of EPA's strategic partnership with coastal states, the Gulf Ecology
Division held a National Coastal Assessment workshop Jan. 29-30, 2002 in Gulf Breeze,
Florida, for 14 representatives from South Carolina, Georgia, Florida, Alabama, and
Mississippi. The National Coastal Assessment (formerly Coastal 2000) is a large-scale,
comprehensive environmental monitoring program that characterizes the Nation's
coastal resources-both estuaries and offshore waters.

      The National Coastal Assessment program is based on EPA's Environmental
Monitoring and Assessment Program that uses Geographic Information System
technology to select sampling sites. Base sites for the first year's monitoring  were
distributed throughout the 24 coastal states and Puerto Rico. Each State or
Commonwealth had at least 35 randomly selected sites that were sampled by its own
resource agency personnel who had already completed the rigorous field sampling
training offered by EPA.

      The workshop transferred EPA-developed technology and expertise to the States
and ensured their complete competency in implementing the program at, for,  and by their
own States.  Topics covered  included the development of assessment questions,
conceptual models, sampling and response designs, quality assurance, data  analysis
and interpretation, information management, and reporting. Similar workshops are being
planned for other States, to cover the entire coastline of the United States.

                         MEETINGS / CONFERENCES

EMAP Symposium 2002 Coming May 7 - 9

      "EMAP Symposium 2002: The Conditions of Our Nation's Streams & Rivers from
the Mountains to the Coasts" will be held May 7 - 9 in Kansas City, Missouri. More than
250 participants from Federal, State, and local governments, tribes, universities, and
nonprofit organizations across the country are expected to attend.

      Co-sponsored by EPA's Office of Research and Development and by the Council
of State Governments, the symposium will be one of the major events of EPA's
Environmental Science Month in May. The principles and procedures developed in
EPA's Environmental Monitoring and Assessment Program (EMAP) will be applied
directly to help States and tribes evaluate the quality of their streams and rivers.
Because EMAP has already been used successfully in the East and a major effort is
currently under way on western streams, it now can be applied to inland surface waters.
Hence the location of the symposium in America's heartland.  Later, EMAP procedures
will be applied to the great rivers of the Midwest-the Mississippi, Missouri, and Ohio.

      Four themes are used to organize the symposium:

            Monitoring Streams and Rivers of the U.S.:  EMAP-West and State Studies

            Using EMAP to Create the Big Picture of Great Rivers

            Integrating 305(b) and 303(d) of the National Water Quality Inventory
            Report: How EMAP Aids in Monitoring and Assessment of State Waters

            Socioeconomic Monitoring and Assessment

       For additional information, visit the symposium Web site at
http://www.csg.org/emap  symposium 2002.htm .


Atlas Provides Scientific Basis for Land Use Planning

      An atlas that charts environmental impacts for different development scenarios
has been completed after an eight-year collaborative research project that brought
together Federal, State, and university scientists to address land-use implications in

      Since 1994, the Pacific Northwest Ecosystem Research  Consortium, an umbrella
organization including the Western Ecology Division  (WED), the University of Oregon,
Oregon State University, and the University of Washington, has been studying Oregon's
Willamette River Basin in  great detail, not just at the  local level, but at the much broader
landscape level.  Their research  is culminating this spring in the publication of a 178-
page atlas that charts differing environmental futures for the basin.  The large-format
atlas,  "Willamette River Basin: Trajectories of Environmental and Ecological Change," is
being published by Oregon State University Press.

      Dr. Joan Baker of WED was the EPA Project Officer; she is also a co-editor of the
atlas.  The information provided in the atlas will allow the region's residents to understand
the implications and consequences of land-use and development decisions.

      The atlas has three sections.  The first covers base data on landforms, water
resources, biotic systems, human population, and  land use and land cover.  The next
section describes three potential alternative futures for this region of 2 million residents,
and makes projections for each alternative to the year 2050, by which time the

 population is expected to double. Finally, there is an extensive section on potentials for
 restoration of the Willamette River.

       Alternative futures involve three different scenarios for development: one opting
 for extensive conservation measures, one pursuing increased rates of development, and
 one following existing state and local development guidelines. The atlas describes the
 likely effects of each of these future landscape changes on stream condition, wildlife, and
 water availability and use, relative both to current and historical conditions in the
 Willamette Basin.

 Dioxin Toxicity Equivalence Factors in Fish and Wildlife Published

       Scientists have  known for years that dioxin is harmful to humans, fish, and other
 wildlife.  The most potent and intensively studied dioxin, TCDD (2,3,7,8-tetrachloro-
 dibenzo-p-dioxin), is used as the basis or reference compound for determining the
 toxicity of other compounds with dioxin-like  properties. A scientific collaboration has
 advanced the development of assessment tools for determining the toxicity of
 environmental chemicals.

      Three NHEERL scientists  (Dr. Philip Cook of the Mid-Continent Ecology Division
and Drs. Michael DeVito and Tala Henry of the Experimental Toxicology Division) were
key contributors to a recently completed 385-page report on a 1998 workshop on toxicity
equivalence factors (TEFs) used  to compare the toxicity levels of various dioxin-like
compounds. The report, "Workshop Report  on the Application of 2,3,7,8-TCDD Toxicity
Equivalence Factors to Fish and Wildlife" (EPA/630/R-01/002) is available on the Web at
http://www.epa.gov/ncea/raf/rafpub.htm .

      A TEF estimates the potency of an individual polychlorinated dioxin, furan, or
biphenyl to cause dioxin-like effects.  The publication is part of a broader effort of EPA's

 Risk Assessment Forum on how to assess ecological risks posed by mixtures of dioxin-
like compounds to fish and wildlife.

      Sponsored by EPA and the Department of Interior, the workshop was a major step
in jointly addressing the possibility of applying TEFs to the Great Lakes Water Quality
Guidance. The use of TEFs and the toxicity equivalence approach in ecological risk
assessment was  strongly endorsed by workshop participants.  As a result, the NHEERL
authors now work with other scientists from EPA's Regional Offices and Risk Assessment
Forum, the U.S. Fish and Wildlife Service, and the U.S. Geological Survey to address
issues with multi-disciplinary and cross-program complexities in more depth than in the
report. This work is part of an ongoing effort by the forum to develop a framework for
applying the toxicity equivalence methodology in ecological risk assessment.