21ST ANNUAL MEETING
PROGRAM SCHEDULE AND ABSTRACTS
2004 Theme:
Toxicology
in
Third World Settings
MT. BACHELOR VILLAGE RESORT CONFERENCE CENTER
BEND, OREGON
SEPTEMBER 17-19, 2004
Co-sponsors:
OHSU Center for Research on
Occupational and Environmental Toxicology (CROET)
and
Third World Medical Research Foundation
(TWMRF)
20 Years
TOXICOLOGY IN THIRD WORLD SETTINGS
Hemi-inattention syndrome is a remarkable, stroke-related neuropsychological disorder in which
the brain attends to only one side of the body and the environment. Patients have an intuitive
knowledge that the other side of their world exists, but it has no meaningful reality in their daily
lives. Patients with this type of unilateral neglect syndrome actually have to turn around to see
and appreciate the other side of their world. Many western biomedical scientists have another
type of hemi-inattention syndrome: we are vaguely conscious of different occupational and
environmental conditions in less- and least-developed countries, but the concept plays no role in
our daily professional practice.
This year’s meeting seeks to treat our collective hemi-inattention syndrome by highlighting the
challenges of toxicological research, education and practice in third world settings, including
sub-Saharan Africa. Abject poverty, widespread infection, undernutrition, tainted water, and
global parasitism, are some of the many environmental threats that dominate life in the leastdeveloped countries on the African continent. Child labor, gross occupational exposures,
inadequate sanitation, and pesticide overuse, characterize rapidly industrializing less-developed
regions of Asia and Central and South America. The 2004 PANWAT program poses this
question: Are there professional opportunities for research, discovery and contribution in these
diverse third world settings?
Biomedical scientists with diverse backgrounds and hands-on experience in developing countries
will inform our members and motivate students and mentors alike. The PANWAT Executive and
Membership wish to thank the many invited speakers who are addressing this challenging
subject. Particular thanks go to our keynote speakers Dr. Terri Damstra from the World Health
Organization and Valerie Palmer from Third World Medical Research Foundation (TWMRF),
which is celebrating its 20th year of research and education on neglected nutritional and toxic
disorders. They and other speakers will provide the background for a roundtable discussion on
the question posed above.
I would like to add a special note of appreciation to my assistant Rodger whose superb
organizational skills have made this year’s meeting possible. He and I have enjoyed working
closely with PANWAT’s Executive Committee (especially Carin Thomas) to bring the 2004
program and meeting to fruition. Thanks are also extended to Mount Bachelor Village Resort
and CROET at OHSU for organizational (Rodger Metheny) and audiovisual support (Dan
Austin). This year’s program would not have been possible without sponsorship from OHSU and
from TWMRF.
Peter Spencer
PANWAT President
2004-2005
Program and Meeting Schedule
(Conference Center)
Friday, September 17th:
6:00 to 9:00 pm
6:00 to 9:00 pm
6:00 to 9:00 pm
Meeting registration (Spring Village Canyon Foyer East)
Submit platform presentations to AV specialist Dan Austin (Winter’s Hope B)
PANWAT Officers meeting, Café Rosemary
1110 NW Newport Avenue
Bend, Oregon
Ph: 541-317-0276
Saturday, September 18th:
TOXICOLOGY IN THIRD WORLD SETTINGS:
Mini-Symposium: Defining the Challenges in Perspective
Guest speakers* sponsored by:
Center for Research on Occupational and Environmental Toxicology (CROET)
at Oregon Health and Science University (OHSU)
Poster set up (Winter’s Hope B)
Meeting registration (Foyer)
Breakfast (Winter’s Hope A)
7:00 to 8:25 am
7:00 to 8:25 am
7:45 to 8:30 am
I. Invited Speaker Session (Winter’s Hope B)
Introduction: Peter Spencer, President, PANWAT
8:40 to 9:20 am
KEYNOTE: *Terri Damstra, World Health Organization
“Environmental threats to human health: A global perspective”
9:20 to 10:00 am
*Thomas Irungu, Mayo Clinic, Rochester, Minnesota
“Toxicologic research in Africa: Moving from anecdotes to epidemiology”
10:00 to 10:20 am
Break (Exhibitor Hall)
II. Invited Speaker Session (Winter’s Hope B)
Introduction: Rosita Rodriguez-Proteau, President Elect, PANWAT
10:20 to 11:00 am
*Rhian Cope, Oregon State University
“UV radiation and Buruli ulcer disease”
11:00 to 11:40 am
*Hoffman M. Lantum, Eastman Kodak, Rochester, NY
“The ‘heavy weight’ of lead in 21st Century developing nations”
11:40 to 12:20 pm
Lilian Calderon-Garciduenas, University of Montana
“Brain inflammation and Alzheimer’s-like pathology in Third World individuals
exposed to severe air pollution”
12:20 to 1:25 pm
Lunch (Winter’s Hope A)
Saturday, September 18th:
III. Student and Post-Doc Speaker Session (Winter’s Hope B)
Introduction: Castle Funatake, Student Representative, PANWAT
1:30 to 1:55 pm
Stephanie Smith-Roe, Oregon State University
“Mlh1-deficient mice are hypersensitive to Phlp-induced mutation and aberrant
crypt foci: Evidence for differential processing of PhlP adduct mispairs by the
mismatch repair pathway”
1:55 to 2:20 pm
Priscilla Fernandes, Oregon Health & Science University
“Mammalian cell mutagenesis and in vitro mutagenic replication bypass of DNA
containing site-specific butadiene-derived N3-RS uracil adducts”
2:20 to 2:45 pm
Severine Navarro, University of Montana
“Consequences of Aryl hydrocarbon Receptor (AhR)-mediated signaling in
dendritic cells”
2:45 to 3:10 pm
Mark Reimers, Oregon State University
“Two zebrafish alcohol dehydrogenases sharing common ancestry and functional
characteristics with mammalian class I and III genes”
3:10 to 3:30 pm
Break (Exhibitor Hall)
IV. Student and Post-Doc Speaker Session (Winter’s Hope B)
Introduction: Cecile Krejsa, Councilor, PANWAT
3:30 to 3:55 pm
Dongren Yang, Oregon Health & Science University
“Polychlorinated biphenyls (PCBs) impair cognitive function via modulation of
dendritic growth and maturation”
3:55 to 4:20 pm
Ava Rhule, University of Montana
“The immunomodulatory effects of notoginseng on cultured phagocytic cells”
4:20 to 4:45 pm
Lisa M. Smith, University of Washington
“Response of an ApoE-/- mouse model to Seattle PM”
4:45 to 7:00 pm
Reception and Poster Session (Winter’s Hope A)
Cycled video presentations (see page 37):
Courtesy of Third World Medical Research Foundation.
“The Poison that Waits” (Guam, Japan, New Guinea)
“Konzo” (sub-Saharan Africa)
“Seychelles Paraplegia” (Indian Ocean Island)
Sunday, September 19th:
TOXICOLOGY IN THIRD WORLD SETTINGS:
Mini-Symposium: Toxic-Nutritional Disorders of the Brain
Lunch & guest speakers* sponsored by:
Third World Medical Research Foundation (TWMRF)
8:15 to 8:55 am
Breakfast (Winter’s Hope A)
V. Invited Speaker Session (Winter’s Hope B)
Introduction: Peter Spencer, President, PANWAT
9:10 to 9:40 am
KEYNOTE: *Valerie Palmer, Third World Medical Research Foundation and
Oregon Health & Science University
“Lathyrus and lathyrism: Highlighting 20 years of research and education on
neglected disorders”
9:40 to 10:20 am
*D. Desire Tshala-Katumbay, University of Kinshasa, Democratic Republic of
Congo, and Oregon Health & Science University and TWMRF
“Cassava and Konzo in the Sub-saharan Africa”
10:20 to 11:10 am
Peter S. Spencer, Oregon Health & Science University and TWMRF
“Cycad and Western Pacific Amyotrophic Lateral Sclerosis ParkinsonismDementia Complex (ALS-PDC)”
11:15 am
Pick-up Box Lunch (West Deck)
11:30 to 2:00 pm
Roundtable Discussion: Opportunities for Research, Discovery and
Contribution
Invited speakers with John Tor Agbidye and Richard Pleus
2:00 to 2:15 pm
Graduate Student Award Presentations
Platform Presentations ($200 first place, $100 second place)
Poster Presentations ($150 first place, $75 second place)
General Information
Platform and Poster Presentations:
Platform presentations, in PC or Mac format, can be e-mailed to Rodger Metheny (methenyr@ohsu.edu)
by September 14th or submitted (CD or Zip disk) in person to the AV specialist at the conference center
(meeting site) between 6 and 9 pm on Friday, September 17 (Winter’s Hope B).
Poster presentations: Posters should be 4’ x 6’ in size. Push-pins will be available at the meeting to
attach each poster to the conference center wall.
Housing:
Attendees must make housing arrangements themselves. Attendees are encouraged to stay at the Mount
Bachelor Village Resort and Conference Center. You may view the facilities including a java script of
the Ski House Loft room on their website at http://www.mtbachelorvillage.com. Note that the
PANWAT rates quoted below are significantly reduced from regular rates. Three types of rooms are
available for us at the resort:
1. Ski House Loft room, two queen beds in separate rooms and one pull out couch in the living room,
$138.
View: http://vtours.matrixdm.com/jbmarketing/ipix/virtualtourold.cfm?tourname=mt.bcondo&view=2
2. Ski House Room (one bedroom), one queen in bedroom and one pull out couch in living room, $89
3. River Ridge Executive Room, one queen, $69.
To make reservations call Cindy Jordan at 1-800-452-9846. Be sure to mention PANWAT.
Dress:
Casual
Food:
Breakfast (on Saturday and Sunday) and Lunch (Saturday) will be served in the conference center.
Sunday lunch will be sponsored by Third World Medical Research Foundation. Everyone is on their
own for Saturday dinner. The resort restaurant is open for dinner and there are many fantastic
restaurants in the town of Bend, only a few miles away from the resort (information available at the
Resort’s Front Desk).
For additional information on the meeting, contact Rodger Metheny (methenyr@ohsu.edu: 503-4942517) or Carin Thomas (cthomas@cwu.edu; 509-963-2815).
For more information on PANWAT, including membership registration forms, see
http://www.toxicology.org/memberservices/regionalchapter/panwat/home.htm.
2004-05 PANWAT Officers
President
Peter Spencer
OHSU, Center for Research on
Occup. & Env. Tox. (CROET)
3181 SW Sam Jackson Park Road
CROET L606
Portland, OR 97239
Ph: (503) 494-4273
Fx: (503) 494-4278
spencer@ohsu.edu
Vice President
Rosita Rodriguez - Proteau
Oregon State University
Pharmaceutical Sciences
203 Pharmacy Building
Corvallis, OR 97331
Ph: (541) 737-5786
Fx: (541) 737-3999
rosita.rodriguez@orst.edu
Vice President-Elect
Andrij Holian
University of Montana
Center For Env. Health Sciences
153 Skaggs Building
32 Campus Drive
Missoula, MT 59812
Ph: (406) 243-4018
Fx: (406) 243-2807
aholian@selway.umt.edu
Secretary/Treasurer
Carin Thomas
Central Washington University
Dept of Chemistry
MS 7539
Ellensburg, WA 98926-7539
Ph: (509) 963-2815
Fx: (509) 963-1050
cthomas@cwu.edu
Councilor: (2 year term)
Cecile Krejsa
Pre-clinical Development
ZymoGenetics, Inc.
1201 Eastlake Ave. E
Seattle, WA 98102
(206) 515-4974
krejsac.zgi.com
Councilor: (1 year term)
David M. Shepherd, Ph.D.
Center for Env. Health Sciences
Skaggs Building, Room 58
32 Campus Drive
University of Montana
Missoula, MT 59812
Ph: (406) 243-2224
Fx: (406) 243-2807
david.shepherd@umontana.edu
Past President
Marc W. Fariss, Ph.D.
College of Pharmacy
Washington State University
P.O. Box 656534
Pullman, WA 99164-6534
Ph: (509) 335-4689
fariss@mail.wsu.edu
Student Representative
Castle Funatake
Oregon State University
Environmental and Molecular Toxicology
1007 Agricultural Life Sciences
Corvallis, OR 97331
Ph: (541) 737-2096
Fx: (541) 737-0497
funatakc@onid.orst.edu
ABSTRACTS
INVITED SPEAKERS
TOXICOLOGY
IN
THIRD WORLD SETTINGS
20 Years
KEYNOTE LECTURE
Environmental threats to human health: A global perspective.
Terri Damstra, PhD.
Team Leader, Interregional Research Unit
International Program on Chemical Safety
World Health Organization (WHO)
Research Triangle Park, NC
Toxicologic research in Africa: Moving from anecdotes to epidemiology
Thomas Irungu, MB, ChB, MPH,
Family Medicine/Preventive Medicine, Mayo Clinic, Rochester, NY
Decades of neglect with regards to the exposure of the majority of its population to toxic
materials makes Africa a goldmine for the toxicologist. However, data that gives an
accurate indication of this situation, and especially its effect on the population are
unavailable. The number of published studies is far outweighed by anecdotes in the press,
oral literature and a few small series held by a handful of poison advisory agencies.
This presentation emphasizes the need and importance of collecting epidemiological data
for toxicologic research in Africa. Environmental conditions such as unavailability of clean
water and sanitation, high prevalence of infectious diseases, poverty, lack of appropriate
technology as well as inadequate policy development and implementation, make research
in this setting challenging. Some of these factors are explored.
UV radiation and Buruli ulcer disease
Cope RB, Bermudez L
College of Veterinary Medicine, Oregon State University, Corvallis, OR
Buruli ulcer disease (BUD) is a significant emerging human skin disease caused by
Mycobacterium ulcerans (MU) infection. Currently, there is no effective in vivo
antimicrobial treatment for BUD. UV exposure results in increased severity of M.
ulcerans infection and suppression of delayed hypersensitivity (DTH) responses of
infected animals to MU antigens. The mechanism of this effect has not been previously
examined. Thus, the aim of this study is to test the hypothesis that topical exposure to cisurocanic acid (cUCA), one of the initiators of the photoimmunosuppressive cascade, would
result in an increased severity of MU infection in a hairless guinea pig model of BUD.
Three groups of 6 animals were exposed to 0.1, 0.5 or 1 mg of cUCA per day for 3
consecutive days. Three groups of 6 animals were exposed to 0.1, 0.5 or 1 mg of transurocanic acid (tUCA) per day for 3 consecutive days. Six animals were included in the
vehicle-treatment-only negative control group, and a group of 8 animals received no
topical treatments (negative control group). One day following the final topical treatment,
all animals were intradermally infected with an ulcerigenic dose (108 CFU) of MU on the
right flank and intradermally injected with vehicle on the left flank. Within 3 days postinfection, typical BUD nodules were present and, by day 5, classical buruli ulcers were
present. By 5 days following infection, and for the remainder of the experiment, all
cUCA-treated animals had significantly larger (p < 0.0001) overall BUD lesions compared
with all other treatment groups. There were no significant differences (p < 0.4) in lesion
size between the tUCA, vehicle-treatment-only and, no-treatment groups. cUCA exposure
also resulted in modulation of the DTH responses following MU antigen challenge at 21
days post-infection. These studies imply a role for cUCA-susceptible immune pathways in
the pathogenesis of BUD.
The ‘heavy weight’ of lead in 21st Century developing nations
Hoffman M. Lantum, MD, PhD
President, African Society for Toxicological Sciences, Toxicologist, Eastman Kodak
Company, Rochester, NY
Environmental exposure to lead has been recognized for more than 100 years as a public
health hazard. The current accepted threshold for toxicity is associated with blood lead
levels (BLL) ≥10 g/dL. Elevated BLLs are associated with neurological, cardiovascular,
bone, hematologic, reproductive and renal disorders, all of which collectively represent
approximately 15% of the total disease burden in available estimates for sub-Saharan
Africa. The long-term effects of lead are particularly severe in developing children
because of the decline in IQ of 7.4 points for every 10 g/dL increase in BLL. Numerous
studies in Africa show 34%-78% of children with elevated BLL, with more than 5% in
some cases having BLL >40 g/dL, a concentration associated with severe neurological
disorders. Average BLLs in most African countries are higher than those of children in
Europe and the United States of America. Elevated BLLs are predominant in the urban
dwellings compared with rural townships. Key environmental risk factors for elevated
BLL are contaminated soil and dust in the urban environment, largely secondary to
automobiles using leaded gasoline. Gasoline sold in most African countries contains 0.50.8 g/l lead, the highest in the world. Of the total amount of lead released into the
environment in Nigeria, approximately 96.7% comes from the combustion of fossil fuels.
Numerous studies in Africa have also shown that one of the strongest indicators of
childhood exposure to lead is the family either owning a car or living next to a tarred road.
In addition to automotive and industrial sources, cottage industries and the burning of
paper products, discarded rubber, battery casings and painted woods for cooking and
heating represent additional hazards to individual households. Lead paint, lead solder and
lead cosmetics are unregulated in most low-income countries in Africa, South America and
Asia. Available data indicate that the average lifetime exposure to lead in low-income
countries results in a greater burden of disease in children than nutritional deficiencies,
HIV, tuberculosis, STDs, cardiovascular diseases, war, and traffic accidents. Only
diarrhea, respiratory infections, malaria, perinatal conditions, and measles rank above the
lead-linked burden. The data presented point to childhood lead poisoning as a major
environmental health problem in urban areas of Africa that require urgent intervention,
notably a ban on leaded gasoline in African countries.
Brain inflammation and Alzheimer's-like pathology in Third World individuals
exposed to severe air pollution
Lilian Calderon-Garciduenas, MD, PhD
Center for Environmental Health Sciences, School of Pharmacy and Allied Health
Sciences, University of Montana, Missoula, MT.
Air pollution is a complex mixture of gases (e.g. ozone), particulate matter, and organic
compounds present in outdoor and indoor air. Dogs exposed to severe air pollution exhibit
chronic brain inflammation and acceleration of Alzheimer's-like pathology, suggesting that
the brain is adversely affected by pollutants. We investigated whether residency in Third
World cities with high levels of air pollution is associated with human brain pathology.
Expression of cyclooxygenase-2 (COX2), an inflammatory mediator, and accumulation of
the 42-amino acid form of beta amyloid, a cause of neuronal dysfunction, were measured
in autopsy brain tissues of cognitively and neurologically intact lifelong residents of cities
having low (n:9) or high (n:10) levels of air pollution. Genomic DNA
apurinic/apyrimidinic sites, nuclear factor-kappaB activation and apolipoprotein E
genotype were also evaluated. Residents of cities with severe air pollution had significantly
higher COX2 expression in frontal cortex and hippocampus and greater neuronal and
astrocytic accumulation of beta amyloid 42 compared to residents in low-air-pollution
cities. Increased COX2 expression and beta amyloid 42 accumulation was also observed in
the olfactory bulb. These findings suggest that exposure to severe air pollution is
associated with brain inflammation and beta amyloid 42 accumulation, two causes of
neuronal dysfunction that precede the appearance of
neuritic plaques and neurofibrillary tangles -- hallmarks of Alzheimer's disease.
Lathyrus and lathyrism: Highlighting 20 years of research and education on
neglected disorders
Valerie S. Palmer
Third World Medical Research Foundation (TWMRF), Portland, OR; Department of
Neurology, School of Medicine, and Center for Research on Occupational and
Environmental Toxicology, Oregon Health & Science University, Portland, OR
(palmerv@ohsu.edu)
TWMRF was formed in 1984 as a very small, non-profit organization designed to sponsor
and conduct research and education on neglected nutritional and toxic disorders prevalent
in least-developed countries (1). The goal was to encourage innovative, low-budget,
multidisciplinary research that would increase understanding of important but little-known
diseases both to benefit affected populations and inform the broad biomedical community.
One example was the discovery among Seychellois of the first cases in Africa of HTLV-1associated spastic paraparesis, a crippling disease that previously had been proposed as a
form of chronic mercury intoxication arising from fish consumption. This study,
performed by an international biomedical team at a cost of less than twenty-five thousand
dollars, was well received by a small country that saw the mercury link as a major threat to
the tourism industry on which its people depended. A much larger initiative focused on
the development of a safe strain of the grass pea (Lathyrus sativus), a hardy and nutritious
but potentially neurotoxic legume that can cause another crippling disease known as
lathyrism (2). While lathyrism has affected human or animal populations on at least four
continents, today human cases are present in Africa (Ethiopia, Eritrea), on the Indian
subcontinent (Bangladesh, India, Nepal), in northwest China, and in parts of Europe
(Spain). TWMRF formed an International Network for the Improvement of Lathyrus
sativus and Eradication of Lathyrism (INILSEL) which, through conferences and
newsletters (3), brought together scientists of widely disparate disciplines to accelerate
understanding of the relationship between the amino acid neurotoxin (BOAA: beta-Noxalylamino-L-alanine) and the human disease, with the long-term goal of promoting
development and use of safe, low-BOAA strains of grass pea for food and fodder. Lowtoxin strains have since been developed in several laboratories; their potential use and
safety will be examined at an international conference held in Syria in October 2004 under
the auspices of the International Center for Agricultural Research in the Dry Areas
(ICARDA) (4). While TWMRF-sponsored scientists in Bangladesh and Ethiopia played
an important role in defining the agricultural, nutritional and biomedical challenges
presented by the grass pea and lathyrism, perhaps the more significant accomplishment
was to focus worldwide attention on this neglected neurotoxic disease and to promote
research on the development of a safe foodstuff for poor people living in areas subject to
drought and water-logging.
1. http://www.twmrf.com/
2. Kaul AK, Combes (1986) Lathyrus and Lathyrism. TWMRF, NY, 334p; Spencer PS, Grass
Pea: Threat and Promise. TWMRF, NY, 245p; Abegaz BM, T-Haimanot R, Palmer VS, Spencer
PS (1994) Nutrition, Neurotoxins & Lathyrism: The ODAP Challenge. TWMRF, NY, 139p.
3. http://www.clima.uwa.edu.au/lathyrus/
4. http://www.icarda.cgiar.org/publications/donors/ethiopia/g%5Fgrasspea.htm
Cassava and Konzo in the sub-Saharan Africa
D. Tshala-Katumbay, MD, PhD
Kinshasa University, Democratic Republic of Congo, Department of Neurology, School of
Medicine and Center for Research on Occupational and Environmental Toxicology,
Oregon Health & Science University, Portland, OR (tshalad@ohsu.edu)
Cassava (Manihot esculenta Crantz) is the prime source of dietary energy for over 500
million people dwelling in the tropics. Outside the tropics, the trade of cassava is limited to
animal feed, starch, and tapioca flour that is used as an ingredient in puddings and sauces.
Throughout Africa, bitter varieties of cassava are much more common than the sweet ones.
Bitter varieties contain large amounts of the cyanogenic glucosides, mainly linamarin (2hydroxyisobutyronitrile-ß-D-glucoside) and to a lesser extent lotaustralin. They are
preferred in many areas with low agricultural suitability for many reasons including:
higher yields, resistance to drought and insects, and protection against predators and
thieves. During traditional processing methods to detoxify cassava (e.g., fermentation or
soaking in water, sun-drying and pounding, depending on the expected final product), the
cyanogenic glucosides break down into cyanohydrins and hydrogen cyanide. Incomplete
detoxification results in exposure to both cyanogenic compounds and their metabolites in
the human body. This occurs during food shortages in the community, as in time of
drought, intense commercialization, or war, when household members are forced to reduce
cassava-processing time. The combination of cyanogenic exposure and low dietary intake
of sulphur amino acids is associated with adverse health effects, in particular spastic
paraparesis (konzo) and/or thyroid dysfunction. Potential neurotoxic candidates in the
pathogenesis of konzo are linamarin, cyanide, cyanate, thiocyanate, and 2iminothiazolidine-4-carboxylic acid. Biomarkers of exposure to cyanogenic compounds
include increased urinary excretion of thiocyanate and development of subtle neurological
symptoms such as brisk tendon reflexes and ankle clonus. Clinical studies show that the
main neurological picture is an acute onset of a lathyrism-like spastic paraparesis, or a
tetraparesis with speech and swallowing problems in severely affected subjects.
Interestingly, this distinct clinical picture is well known by non-health professionals in
konzo-affected areas (Democratic Republic of Congo, Mozambique, Tanzania, Central
African Republic, Cameroon, Uganda, and Angola). The disease mainly affects children
and women of childbearing age. Extensive electroneurophysiological investigations have
demonstrated prominent dysfunction of corticomotoneurons and motor descending
pathways in most subjects affected by konzo. While there is no animal model of konzo,
epidemiological studies consistently show a compelling association between the
occurrence of disease and consumption of high cyanogen-containing cassava (occurring in
populations under severe agroecological crisis). Konzo is a preventable crippling
neurotoxic disease.
Cycad and Western Pacific Amyotrophic Lateral Sclerosis and ParkinsonismDementia Complex (ALS-PDC)
Peter S. Spencer, PhD, FRCPath.
Center for Research on Occupational and Environmental Toxicology, Oregon Health &
Science University, Portland, OR (spencer@ohsu.edu)
A major medical mystery began 50 years ago when United States forces took over Guam
and the medical community discovered an unprecedented prevalence of motor neuron
disease (ALS, lytico) and later parkinsonism with dementia (P-D, bodig) among the
indigenous Chamorro population of this and the nearby island of Rota. By 1960, it was
accepted that ALS-PDC is a single, non-transmissible neurodegenerative disorder with
important implications for understanding the etiology and pathogenesis of lookalike
disorders worldwide. This familial disease was considered an inherited condition until
sequential epidemiological studies spanning decades revealed a declining disease
prevalence, an increase in the age of onset, and an evolution in the clinical presentation.
Discovery of two other similar high-incidence disease foci among residents of Kii
Peninsula, Honshu Island, Japan, and Auyu and Jaqai linguistic groups of West Papua,
Indonesia, stimulated research to identify culpable environmental factors common to all
three affected populations. One prominent but largely discounted hypothesis suggests that
low calcium content of food and water triggers a parathyroid hormone response that
promotes gastrointestinal absorption of bystander neurotoxic metals such as manganese
and aluminum. By contrast, Chamorro folklore suggests that lytico is causally related to
eating too much fadang, the seed of the cycad plant (Cycas spp.) that was traditionally
used for food and medicine on Guam. Field research part supported by the Third World
Medical Research Foundation revealed common use of cycad seed and links to ALS in all
three western Pacific disease foci. More recently, others have shown that Chamorros
traditionally consumed flying foxes that feed on cycad seed and bioaccumulate cycad
chemicals. While cycads like other plants contain a plethora of substances, two have been
of special research interest: (1) cycasin, the glycone of methylazoxymethanol (MAM), a
potent DNA-damaging agent, hepatotoxin and developmental neurotoxin, the
concentration of which in Chamorro-prepared cycad flour is very strongly correlated with
historical disease incidence; (2) beta-N-methylamino-L-alanine (BMAA), a neurotoxic
amino acid that reportedly bioaccumulates in flying foxes and which in orally dosed
primates produces motor-system abnormalities reminiscent of the human disease. While
the disappearing use of cycad materials for food (Guam) and/or medicine (Guam,
Indonesia, Japan) correlates with declining disease prevalence in all three population
groups, the etiologic role of this plant in ALS/PDC is unproven. Whatever factor is
culpable, a key challenge is to illuminate mechanisms by which an agent can induce a
progressive neurodegenerative disease that may appear clinically years or decades after the
last known exposure to the responsible environment.
Toxicology in the Africa setting, promoting human health and safe environments
through research and education: Emerging opportunities and challenges
John Tor-Agbidye, DVM, PhD
Immediate-Past President, African Society for Toxicological Sciences (ASTS)
Stewartsville, NJ
The western world can count on its pool of experts in the area of toxicological sciences
with existing data that serve as a reference guide in the formulation of health policies.
However, in Africa, there is a low distribution of manpower with this expertise and lack of
data to guide in the formulation and implementation of such policies. Therefore, the need
for development and training of personnel in African universities and allied research
institutions cannot be overemphasized. The African Society for Toxicological Sciences
[(ASTS) www. africansocietyfor toxicologicalsciences.org] is a non-profit organization of
scientists in academia, industry and governmental organizations from around the world
interested in promoting the acquisition and utilization of knowledge in the toxicological
sciences relevant to the continent of Africa. The founding members of the organization
were mostly those from the continent of Africa living and working in the USA and Europe.
The members believe that charity begins at home and they have to give back their expertise
to Africa. ASTS membership is open to individuals and institutions around the world
interested in toxicological issues facing the African continent. ASTS has set for itself a
noble goal, that of promoting the acquisition and utilization of knowledge in toxicological
sciences relevant to the continent of Africa and to facilitate the exchange of information
among its members as well as among investigators of other scientific disciplines. We also
encourage cooperative research in environmental health with established scientists in the
West. ASTS has received major support from the National Institute for Environmental
Health Sciences (NIEHS) for its continent-wide activities in partnership with the Third
World Medical Research Foundation. The NIEHS mission of understanding how
individual susceptibility, environmental exposures, and behavior influence health and
disease is not limited to concerns within the United States. Congruent with that mission is
the goal to promote the capacity for environmental health sciences research in developing
countries. Through such efforts, countries make educated decisions as they formulate their
own environmental health policies. Through the ASTS and country/region-based scientists
in environmental toxicology, environmental epidemiology, and public health, we hope to
continue toward our goal of a safer world for ourselves and for our children.
Research, Discovery, and Contribution: Professional Experience in the Republic of
South Africa
Richard C. Pleus, PhD
Intertox, Inc.
Department of Pharmacology, University of Nebraska Medical Center, NE
Center for Environmental Toxicology, University of Nebraska, NE
Following the dismantling of apartheid in the Republic of South Africa during 1994 and
1995, and the newly approved national constitution in May of 1996, there was revived
vigor regarding problems of environmental pollution and human health. I was invited to
South Africa to assist in establishing a program in risk assessment. During my three visits
to South Africa between 1997 and 1998 I participated in technical discussions related to
risk assessment and toxicology with governmental and private industry representatives.
The discussions focused on land, water, and air legislation and the benefits of risk-based
legislation. I provided courses in human health risk assessment and toxicology for staff of
the Council on Science and Research (CSIR) to introduce multi-pathway risk assessment
as a means for evaluating potential chemical exposures associated with various industrial
activities in South Africa. I was asked to review a multi-pathway human health risk
assessment for the CSIR. The focus of the risk assessment was to assess the health impact
to residents near the waste incinerator facility for the Baragwanath medical hospital in
Johannesburg (Soweto). This risk assessment was the first of its kind in the Republic of
South Africa. Providing professional services as a toxicologist in South Africa was a
valuable informational experience that has formed much of my practice today. I learned
and recognized the importance of basic public health measures as the foundation for
protecting human health and I understood how fundamental economic development can
improve human health.
ABSTRACTS
STUDENT PRESENTATIONS
FREE COMMUNICATIONS
Mlh1-deficient mice are hypersensitive to PhIP-induced mutation and aberrant
crypt foci: Evidence for differential processing of PhIP adduct mispairs by the
mismatch repair pathway
Smith-Roe, SL, Crain SS, Buermeyer, AB.
Department of Environmental and Molecular Toxicology, Oregon State University,
Corvallis, OR
Disruption of the DNA mismatch repair (MMR) pathway results in elevated mutation rates
and inappropriate survival of cells bearing DNA damage. Hereditary nonpolyposis
colorectal cancer (HNPCC) is an early onset predisposition for internal cancers caused by
deficiency for one of several genes involved in MMR. The extent to which environmental
factors influence the etiology of HNPCC is unknown. 2-amino-1-methyl-6-phenylimidazo
[4,5-b] pyridine (PhIP) is a mutagen and carcinogen present in cooked meat that, when
metabolized, forms DNA adducts. To determine whether PhIP represents a cancer risk to
individuals with MMR-deficiency, Mlh1-/- mice carrying the cII transgene were exposed to
PhIP via i.p. injection. PhIP induced 3-fold more mutations in the colons of Mlh1-/- mice
compared to their wildtype littermates, suggesting that MMR normally suppresses about
70% of potential PhIP-induced mutations. Analysis of mutational spectra in the colon
indicated that Mlh1-/- mice were hypersensitive to -1 frameshifts, G/C to A/T transitions,
and G/C to C/G transversions, whereas G/C to T/A transversions were induced similarly in
both wildtype and Mlh1-/- mice. In cancer studies, PhIP-treated Mlh1-/- mice were
hypersensitive to induction of colonic aberrant crypt foci. In contrast, adenomas of the
small intestine were not induced, despite a similar induction of mutation in the small
intestine as in the colon. Cell-turnover studies of colon and small intestine are in progress
to determine the importance of Mlh1-dependent apoptosis in PhIP-induced
carcinogenicity. These data are consistent with the hypothesis that PhIP exposure
increases mutagenesis and carcinogenesis in Mlh1-/- mice, and support further evaluation of
the risk that consumption of heterocyclic amines may impart to MMR-deficient
individuals.
Mammalian cell mutagenesis and in vitro mutagenic replication bypass of DNA
containing site-specific butadiene-derived N3-RS uracil adducts
Priscilla H. Fernandes1, Richard P. Hodge2, and R. Stephen Lloyd1
1
Center for Research in Occupational and Environmental Toxicology, Oregon Health &
Science University, Portland, OR, 2Sealy Center for Molecular Science, University of
Texas Medical Branch, Galveston, TX
1,3-Butadiene (BD) is a high-volume industrial chemical used in the manufacture of tires,
plastics, resins and other synthetic rubber-based goods. This chemical is of crucial
importance in developing countries where permissible exposure limits may go
unmonitored or BD-based products are burnt and not recycled. BD is a known rodent
carcinogen and a probable human carcinogen. The genotoxicity of butadiene is thought to
be mediated through the formation of DNA adducts from its metabolites. While many of
these adducts are either unstable, weakly mutagenic or obtained in low yields, the N3-RS
BD uracil DNA adducts have been recently characterized as highly stable DNA adducts
from reactions of deoxycytidine or calf thymus DNA with epoxybutene, the primary
metabolite of BD. In our studies, the mutagenic potential of these site-specific adducts
were tested in mammalian cells. We found them to be highly miscoding, accounting for
very high frequencies of C to T and C to A mutations, followed by a much lower
percentage of C to G mutations. The N3-RS BD uracil adducts were subsequently tested
for their effect on mammalian replicative polymerases, pol  and pol . Both human pol 
and calf thymus pol  in the presence of PCNA were severely blocked by the adducts in
comparison to an undamaged control. Human translesional DNA polymerases η, ι and κ
were then evaluated for their ability to bypass the N-3 RS BD uracil DNA lesions. They
were found to bypass the N-3 RS BD uracil adducts, albeit at a lower rate than the
undamaged control. Single-nucleotide incorporation experiments indicate preferential
incorporation of T opposite the adduct in the case of all three polymerases, which in turn
provides a rationale for the C to A mutations detected in vivo. Further evaluation of these
polymerases in extension experiments, using four different primers with one of the four
nucleotides opposite the adduct, showed pol  to be the mostly likely candidate in the
bypass of these adducts in vivo.
Consequences of Aryl hydrocarbon Receptor (AhR)-mediated signaling in
dendritic cells
Severine Navarro and David M. Shepherd
Center for Environmental Health Sciences and Department of Biomedical and
Pharmaceutical Sciences, University of Montana, Missoula, MT.
Exposure to the environmental contaminant 2,3,7,8-tetrachlororodibenzo-p-dioxin (TCDD,
dioxin) causes suppressive effects on the immune system. The mechanism(s) of action at
the cellular and molecular levels underlying the potent suppression of the T cell-mediated
immune response is not understood. We hypothesize that TCDD exposure impedes
activation of CD4+ T cells due to a lack of persistent interactions with the dendritic cells
(DC). To test this hypothesis, C57Bl/6 recipient mice were gavaged with vehicle or
TCDD (15 µg/kg), intravenously injected with OT-II/Thy1.1 ovalbumin (OVA) peptidespecific naïve T cells, and subsequently immunized with OVA-loaded DC-enriched spleen
cells. The fate of both the dendritic cells and the antigen-specific T helper cells in the
draining lymph nodes was then evaluated on multiple days after immunization. On days 3,
4 and 6 post-immunization, TCDD-treated mice had significantly decreased numbers of
CD11c+ DC, and on day 6 a greater frequency of these antigen presenting cells stained
positive for propidium iodide suggesting their loss was due to increased cell death.
Furthermore, the ability of the OVA-loaded dendritic cells to induce clonal expansion of
the OT-II/Thy1.1 CD4+ T cells was significantly suppressed on days 4 and 6 in TCDDtreated when compared to the vehicle-treated controls. In addition, the OVA-specific T
cells in dioxin-exposed mice were less activated based on significantly lower expression of
the activation markers CD11a and CD44, and suppressed production of IL-2 and IFN-
following ex-vivo re-stimulation. Additional studies utilizing OVA-loaded DC from Aryl
hydrocarbon receptor wildtype (AhR+/+) and knockout (AhR-/-) mice in the OT-II adoptive
transfer model demonstrated that the TCDD-induced loss of DC was mediated exclusively
via an AhR-dependent process. However, suppression of the OVA-specific CD4+ T cell
clonal expansion in dioxin-treated mice was only partially restored following the
immunization of adoptively transferred mice with OVA-loaded AhR-/- DC. These results
suggest that both the DC and activated T lymphocytes are directly affected following
activation of the AhR.
Two zebrafish alcohol dehydrogenases sharing common ancestry and functional
characteristics with mammalian class I and III genes
Mark J. Reimers 1,3, Mark E. Hahn 2 and Robert L. Tanguay 3
1
Department of Pharmaceutical Sciences, University of Colorado HSC, Denver, CO; 2
Biology Department, Woods Hole Oceanographic Institute, Woods Hole, MA. 3
Department of Environmental and Molecular Toxicology, Oregon State University,
Corvallis, OR.
Ethanol is teratogenic to many vertebrates, and we are using zebrafish as a model system to
determine if there is an association between alcohol dehydrogenase (ADH) activity and
developmental toxicity. To test this hypothesis, it is necessary to determine if zebrafish
express ADH enzymes capable of ethanol metabolism during development. With the
duplication of many genes in zebrafish, it is critical to ascertain the functional
characterization of all identified enzymes. Here we report the isolation and characterization
of two cDNAs encoding zebrafish ADH. Phylogenetic analysis of these zebrafish ADHs
reveals that they are most closely related to the mammalian class 1 ADH enzymes, and we
have named them adh8a and adh8b. Both adh8a and adh8b were genetically mapped to
chromosome 13 approximately 3.6 megabases from each other. At the predicted amino
acid level, adh8a and adh8b are 77% identical to each other, and are 73% and 68%
identical to the human class 1 ADH, respectively. To characterize the functional properties
of ADH8A and ADH8B, the enzymes were recombinantly expressed and purified from
SF-9 insect cells. Kinetic studies with a variety of substrates revealed differential substrate
preferences. Consistent with mammalian class I enzymes, ADH8A metabolizes ethanol,
with a 0.679 mM Km. Conversely, ADH8B does not oxidize ethanol, but does efficiently
metabolize longer-chain primary alcohols greater than 5 carbons and Shydroxymethlyglutathione similar to class III enzymes. ADH8A does not efficiently
metabolize these substrates. Finally, mRNA expression studies indicate that both ADH8A
and ADH8B mRNA are expressed during early development. Together these results
indicate that class I ADH enzymes are conserved in zebrafish, albeit with mixed functional
properties. Supported by NIH grants AA12783, ES03850, and ES00210.
Polychlorinated biphenyls impair cognitive function via modulation of dendritic
growth and maturation
Dongren Yang, Pamela J. Lein
Center for Research on Occupational and Environmental Toxicology, Oregon Health &
Science University, Portland, OR
Polychlorinated biphenyls (PCBs) are persistent, widely occurring environmental
contaminants. Increasing evidence indicates that perinatal exposure to relatively low levels
of PCBs may cause cognitive and behavioral problems in children. However, assessment
of this risk is complicated by large gaps in our understanding of the mechanisms of PCB
developmental neurotoxicity. Additional uncertainties arise from the lack of data regarding
structure-activity relationships in PCB’s developmental neurotoxicity, and the paucity of
appropriate model systems that examine the effects of perinatal PCBs exposure on
cognition and behavior in developing animals. Although it is widely hypothesized that
PCBs disrupt normal brain development, specific developmental events targeted by PCBs
have yet to be identified. To address these issues, we tested: 1) the feasibility of using
juvenile animals to assess the impact of low-level exposures to PCBs on learning and
memory; and 2) the hypothesis that developmental exposure to PCBs alters dendritic
growth and maturation. The Morris water maze was used to evaluate spatial learning and
memory in juvenile (PN24) rats exposed to PCBs throughout gestation and lactation. Our
data indicate that developmental exposure to low levels of the commercial PCB mixture
Aroclor 1254 at 1mg/kg but not 6mg/kg causes subtle but functionally significant changes
in spatial learning and memory. These changes are consistent with the human
epidemiological data and suggest the validity of using the Morris water maze to evaluate
PCB effects on cognitive function in juvenile animals. Real time RT-PCR analysis of
transcripts for dendrite selective proteins showed that Aroclor 1254 treatment has
significant temporal and spatial effects on the expression of RC3/Neurogranin and
spinophilin in the hippocampus and cerebellum of rats. Morphological measurement of the
layer width in the Nissl-stained brain sections revealed significant changes in the molecular
layers of the cerebellum, but not the hippocampus and cortex. This study integrates
analysis of structural alterations with behavior and functional assessments in the whole
developing animal. These results demonstrate a strong link between impaired cognitive
function and changes in the dendritic morphology.
The immunomodulatory effects of notoginseng on cultured phagocytic cells
Ava Rhule1, Severine Navarro2, Jerry Smith1, and David M. Shepherd1,2
1
Department of Biomedical and Pharmaceutical Sciences and 2Center for Environmental
Health Sciences, University of Montana, Missoula, MT
The FDA does not regulate herbal or dietary supplements as drugs although many may
have associated therapeutic effects and toxicities. In this regard, the immunomodulatory
effects of Panax notoginseng on cultured dendritic cells (DC2.4) and macrophages (RAW
264.7) were investigated. Cells were stimulated with 1 g/ml LPS and treated with
notoginseng at 5, 25 and 50 g/ml. Notoginseng inhibited the production of TNF- by
phagocytes in a concentration-dependent manner. Levels of COX-2 and IL-1 mRNA
were also inhibited in RAW264.7 cells. Inhibition of TNF- production by notoginseng at
different treatment time points relative to LPS addition was studied. TNF- production
was inhibited in samples pre-treated with notoginseng for 24 hours or added concurrently
with LPS, but not in samples treated 8 hours after LPS stimulation. The effects of
notoginseng on the expression of the accessory molecules CD40, CD14, CD86 and TLR4
in LPS stimulated and unstimulated cells were analyzed by flow cytometry. CD40
expression was decreased and TLR4 expression was increased in DC2.4 cells while CD86
and CD14 expression were unaffected at 50 g/ml notoginseng exposure. Similar results
were observed in the RAW 264.7 cells. The immunomodulatory roles of Rb1 and Rg1
ginsenosides, the purported bioactive components of our notoginseng extract were
investigated. Both Rb1 and Rg1 inhibited TNF- levels in the RAW264.7 cells following
LPS stimulation, but only the Rg1 inhibited TNF- production by DC2.4 cells.
Notoginseng inhibited TNF- to a greater extent than did either purified compound.
Collectively, these results indicate that notoginseng inhibits activation of RAW264.7 and
DC2.4 cells and suggest that notoginseng possesses immunosuppressive properties in vitro.
Response of an ApoE-/- mouse model to Seattle PM
L. M. Smith, C. Baker and D. L. Luchtel, Ph.D.
Environmental & Occupational Health Sciences, University of Washington, Seattle, WA
Transgenic apolipoprotein E deficient (ApoE-/-) mice were used to study the potential
health effects of Seattle PM2.5. Dose-response studies were done to monitor lung
inflammatory and cardiovascular responses. Methods: Two study protocols were designed
to complement one another. The first used 11-12 mo male ApoE-/- mice exposed to 0, 50,
150, or 400 µg Seattle PM2.5 (collected on teflon filters) in 30 µl saline by oropharyngeal
aspiration (n = 9-11/group) and age/sex/dose matched C57BL6/J controls (n = 4-8/group).
Animals were sacrificed and lung lavages with 0.8 ml sterile saline (x 6 times) were
performed at 6, 24 and 72 hrs post-exposure. Lavage cells were pelleted by centrifugation
and cells were counted using a hemacytometer. Cell counts were normalized to body
weight. The second study utilized 5 ApoE-/- mice aged 17-18 mos implanted with ECG
telemetry devices (DSI, Minneapolis, MN). Two were exposed to sterile saline and 3 to 50
µg Seattle PM2.5. ECG was monitored for 24 hrs prior to and 72 hrs following exposure.
ECG analysis was performed using Dataquest ART software. Power spectra were divided
into very low frequency (0.05-0.4 Hz), low frequency (0.1-1.5 Hz), high frequency (1.5-4.0
Hz) bands and normalized to total spectra. Results: The ApoE-/- mice showed a lower
baseline cell count than the C57 animals. However, the ApoE-/- mice showed an overall
greater response when exposed to PM2.5 vs the C57 mice. Specifically, at 24 hrs post
exposure, the ApoE-/- mice showed a strong dose response trend with significant results at
50 µg. The dose response trend was also present at 72 hrs. A power analysis of the ECG
data obtained at 24 hrs post exposure showed a significant increase in the high frequency
band of PM2.5 exposed mice vs saline controls. Also, at 24 hrs, there was a trend
indicating a decrease in the very low frequency band. Conclusion: Overall, for both
measures, ApoE-/- mice showed a marked response at 24 hrs post exposure. (EPA #R827355-01-0 and NIEHS #P30 ES07033.)
ABSTRACTS
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Seven cases of fatal paraquat poisoning in Portland, Oregon dogs
RB Cope1, RJ Bildfell1, BA Valentine1, KS White1, BJ Cooper1, Andrea Oncken2
1
College of Veterinary Medicine, Oregon State University, 2Dove-Lewis Emergency
Animal Hospital, Portland, OR
Paraquat is one of the few broad-spectrum herbicides available for use in the United States;
however, it is extremely toxic to companion animals when ingested. Despite its restricteduse status, poisoning of dogs and cats remains relatively common. This clinical report
documents a series of chronologically and geographically related cases of presumed
malicious and fatal sub-acute paraquat poisoning in seven dogs in Portland, Oregon. All
animals developed a clinical syndrome by acute gastrointestinal disturbance, renal
compromise and insidiously progressive respiratory failure. Hyperlipasemia and moderate
hypertension were notable feature in five out of the seven cases. Trace levels of paraquat
were demonstrated in the urine of four out of seven animals by gas-liquid
chromatography/mass spectroscopy. Diagnosis in the remaining three cases was made
through a combination of history or exposure, clinical signs and progression and
pulmonary and renal histopathology.
Fatal Yew (Taxus sp.) poisoning in Willamette Valley, Oregon horses
RB Cope1, C Camp2, CV Löhr1
1
College of Veterinary Medicine, Oregon State University, 2Del Oeste Veterinary Hospital,
Eugene, OR.
Despite the well-founded reputation of plants of the genus Taxus as being amongst the
most toxic plants for domestic livestock in the USA, there are surprisingly few published
case reports of yew poisoning in horses. This case report documents two acute fatalities in
horses in the central Willamette valley, Oregon, that were associated with the consumption
of Taxus sp. The predominant features of the intoxication were peracute death, with no
signs of struggling or convulsions, in otherwise fit and well managed adult horses. The
most significant gross necropsy findings were limited to pulmonary congestion and
hemorrhage, suggestive of acute circulatory disturbance. A diagnosis of Taxus sp.
poisoning was confirmed on the basis of a history of potential exposure, by the
identification of yew leaves in the gastric contents of the horses and by the subsequent
identification of yew clippings in the pasture.
Computational model for radiation-induced abnormalities at low doses in the
developing neocortex
NM DeFrank, WC Griffith, JM Gohlke, EM Faustman
Department of Environmental and Occupational Health Sciences, University of
Washington, Seattle, WA.
There is substantial evidence that low-dose radiation exposures (10 to 50cGy) during
gestation can result in permanent neuronal perturbations. The hypothesized mechanisms
underlying these effects include p53-mediated cell-cycle arrest and cell death among
neuronal precursors, leading to alterations in neuron production. Using existing data sets,
we constructed a computational model to describe the relationship between cell cycle
changes, cell death patterns, and mature neuron number at the end of neurogenesis in the
murine neocortex. Model results indicate that radiation-induced stabilization of p53 in
neuronal precursors leads to variable ratios of cells undergoing cell death or DNA repair in
a dose and time dependent manner, with the most drastic effects correspond to times of
rapid proliferation and differentiation. Predictions show a 1% decrease of neuronal
precursor cells per cGy during the most sensitive time window of neurogenesis and about a
0.5% decrease per cGy during other times of neurogenesis. Following DNA repair, other
cells re-enter the cell cycle and exhibit variations in subsequent cycling kinetics, which
further affect final neuron counts. Mouse studies have shown that a single maternal dose
of 20cGy on the 13th day of gestation induced cell death in half of the p53 expressing
neurons and repaired cells experience persistent changes in cycling times. According to
our biologically based model, these events resulted in a quarter reduction of mature
neurons at the end of neurogenesis. Our model provides a method for comparing
differences in absorbed dose, time and duration of exposure, and role of cell signaling
pathways in mediating radiation damage in the neocortex. Supported by the Center for
Children’s Environmental Health Risk Research (R 826886-01-0 and P01 ES09601) and
Department of Energy (DE-FG02-03ER63674).
The immunomodulatory effects of amentoflavone on cultured macrophages
(RAW264.7) and dendritic cells (DC2.4)
Veronica Grijalva1, Severine Navarro2, Ava Rhule3 and David M. Shepherd 1,2,3
1
NSF-EPSCoR Undergraduate Summer Diversity Research Program, 2Center for
Environmental Health Sciences, and 3Department of Biomedical & Pharmaceutical
Sciences, University of Montana, Missoula, MT 59812
Amentoflavone is a biflavonoid ubiquitously found in plants and purported to have antiinflammatory activity. Traditionally, amentoflavone has been used in eastern medicine as
a therapeutic agent to treat advanced cancers and chronic inflammatory diseases; however,
information is lacking on the medicinal and toxic properties of this herb. Therefore, it is
important to investigate its potential effects in order to determine the value and risks that
amentoflavone may pose to human health. In the present study, we observed dosedependent effects of amentoflavone in an in vitro model of inflammation that utilized LPS
to stimulate the production of pro-inflammatory mediators by mouse macrophages (RAW
264.7) and dendritic cells (DC2.4). Each cell line was cultured for 24 hours with or
without LPS (1 µg/mL) and increasing concentrations (5, 25, and 50 g/mL) of
amentoflavone. The production of TNF, IL-1, COX-2, and iNOS was evaluated by
ELISA or RT-PCR. Additionally, expression of the activation markers CD14 and CD40
was evaluated by FACS. Our investigations confirmed previous reports that LPS induces
an inflammatory response in both the macrophage and DC cell lines. Amentoflavone
suppressed the LPS-induced production of TNF in a concentration-dependent manner by
the DC while generating a biphasic response by the macrophages. The LPS-induction of
IL-1 mRNA in both cell lines was suppressed by amentoflavone treatment. In contrast,
COX-2 and iNOS mRNA showed a trend towards being up-regulated by this herbal
extract. Furthermore, no significant alterations were observed in CD40 and CD14
expression in LPS-stimulated RAW264.7 cells treated with amentoflavone. While
amentoflavone can modulate the inflammatory response by phagocytic cells, further
investigations are needed to thoroughly define the immunomodulatory potential of this
herb.
Global gene expression is perturbed by the genotoxin methylazoxymethanol (MAM)
in both the developing cerebral cortex and cerebellum of postnatal mice
G.E. Kisby1*; P. Pattee2; M. Standley2; X. Lu; J. O’Malley2; B. Lin1; J. Muniz1; N.L. Luo2;
D.J. Beardsley2; S. Back2; S.R. Nagalla2
1
CROET, 2Department of Pediatrics, School of Medicine, Oregon Health & Science
University, Portland, OR.
The genotoxin methylazoxymethanol (MAM) has been widely used as a developmental
neurotoxin in various animal models, and its glucoside (i.e., cycasin) is associated with the
prototypical neurodegenerative disorder western Pacific ALS/PDC. The mechanism of
MAM-induced acute or chronic brain injury is poorly understood. We used large-scale
mouse cDNA microarrays (27,648 clones) to study global gene expression changes in the
cerebellum and cerebral cortex to identify the key regulatory targets of MAM during CNS
development. C57BL6 mice (postnatal day 3, PND3) were injected with a sub-lethal dose
of MAM (43 mg/kg, s.c.) and total RNA from the cerebellum and cerebral cortex of PND
4, 8, 15, and 22 mice examined for morphology, DNA damage (N7-methylguanine,
TUNEL labeling), and gene expression. The morphology of the cerebellum and cerebral
cortex of MAM-treated PND4 mice was unremarkable despite an increase in DNA damage
(N7-methylguanine, TUNEL labeling). Gene expression results revealed: 1. MAM
regulates a distinct set of genes in the cortex and cerebellum. 2. Greater than 80% of the
genes influenced by MAM in both brain regions are developmentally regulated. 3. MAM
induced gene expression changes are predominantly early (PND4) in the cerebellum and
delayed (PND 8 and 15) in the corresponding cortex. 4. Persistent changes in gene
expression were observed in both brain regions. 5. Functional classes of molecules
influenced by MAM varied by brain region with a distinct profile of individual
components of the proteasome degradation pathway affected in the cerebellum and DNA
repair pathways in the cortex. These studies demonstrate that the environmental genotoxin
MAM induces gene expression changes in the postnatal cortex as well as the cerebellum
and that these changes are temporal with early and persistent changes occurring in both
immature (cerebellum) and mature (cerebral cortex) brain.
[Supported by NIH grant 1 U19 ES 011384-01 (SN) and DOD grant DAMD17-98-1-8625
(GK)]
Gene expression in methylazoxymethanol (MAM) treated cerebellar neurons is
distinct when compared with other DNA damaging agents
G.E. Kisby1; M. Standley2*; P. Pattee2; J. O’Malley2; J. Muniz1; A. Olivas1; S.R. Nagalla2
1
CROET, Portland, OR, USA, 2Department of Pediatrics, School of Medicine, Oregon
Health & Science University, Portland, OR.
We have shown that DNA damage induced by the alkylating agent methylazoxymethanol
(MAM) is associated with perturbation of global gene expression in the postnatal
cerebellum and cerebral cortex and that the damage precedes the disruption of cerebellar
development (for details see our other poster). Moreover, MAM targeted a large number
of genes that are involved in the development of cerebellar neurons (i.e., granule cells).
The present studies continue our investigation of the influence of MAM on neuronal
development by examining gene expression in mature cultures of granule cells derived
from the cerebellum of C57BL/6J mice. Parallel cultures were also treated with nitrogen
mustard (HN2), a related alkylating agent, to determine if the genotoxin MAM targets a
distinct set of genes. Large-scale mouse cDNA microarrays (27,648 clones) were used to
study gene expression in granule cell cultures that had been treated for 24h with a sublethal concentration of MAM (100 µM) or nitrogen mustard (1.0 µM). After treatment,
DNA from MAM-treated cultures was examined for damage (i.e., N7-methylguanine)
while total RNA from MAM and HN2 treated cultures was examined for gene expression.
Although the viability of MAM-treated cerebellar neurons was unremarkable, we found a
~2-fold increase in DNA damage (N7-methylguanine). Gene expression results revealed:
1. Global gene expression was predominantly up-regulated (~50%) by both genotoxins. 2.
The number of down-regulated genes was ~3-fold greater for HN2 than MAM. 3. MAM
and HN2-regulated a distinct set of genes with minimal overlap (~20%). 4. Only a few
genes were observed to be similarly affected by MAM in both neuronal cultures and the
PND4 cerebral cortex (2 out of 53) or cerebellum (83 out of 603) of mice. 5. Functional
classes of molecules influenced by MAM and HN2 were distinct with MAM having
pronounced effects on cell proliferation, DNA repair, immune response, neurogenesis,
signal transduction, transcription, transport and the ubiquitin pathway while HN2 primarily
influenced apoptosis, hormones, proteosome, and protein synthesis. These studies
demonstrate that MAM induces distinct effects on neuronal gene expression when
compared with other related genotoxins. These differences may account for the variable
response of brain tissue or neurons to different types of DNA damaging agents and explain
the short- and long-term effects of these agents on the CNS.
[Supported by NIH grant 1 U19 ES 011384-01 (SN), 5P42-ES10338-02 (GK) and DOD
grant DAMD17-98-1-8625 (GK)]
In vivo determination of 1,2-diethylbenzene and 1,2-diacetylbenzene in F344 rat
blood
Joel A. Klein1, Allison M. Cartmell2, Richard A. Gies3, Hong Wu3, Karla Thrall3
1
Oregon State University (OSU), Corvallis, OR 97330; 2Gonzaga University
Spokane, WA 99258; 3Pacific Northwest National Laboratory (PNNL)
Richland, WA.
Diethylbenzene (DEB) is a colorless liquid at room temperature used in the production of
divinylbenzene, as a heat transfer fluid, and can be found in gasoline, kerosene, and fuel
oils. The ortho isomer, 1,2-DEB, and its metabolite 1,2-diacetylbenzene (1,2-DAB) have
been shown to have neurotoxic and chromogenic effects on poisoned rats, but 1,2-DAB
had previously only been detected in rat urine, not blood. In this study the toxicokinetics
of 1,2-DEB and 1,2-DAB were investigated in male F344 rat blood after intraperitoneal
injection with 30 mg 1,2-DEB. Amounts of compound in blood were quantified at various
times post injection using a preliminary HPLC/MSD method and the presence of both
substances in the blood was verified using GC/MS. The amount of 1,2-DEB in the blood
was greatest (18 μg) at six hours post injection and 1,2-DAB reached its peak (9.5 μg) after
24 hours. 1,2-DEB was eliminated from the blood by 24 hours post exposure, but 1,2DAB was present even at 48 hours. The HPLC/MSD method appears sufficiently selective
for determination of 1,2-DEB and 1,2-DAB in blood, tissues, and potentially urine. These
data and methods can be used in the development of mathematical models to understand
the potential health effects in humans. (Supported by 1-P42-ES-10338-03 from the
National Institute of Environmental Health Sciences, NIH, with funds from EPA).
Paraquat-induced apoptosis in PC12 cells: a possible link to Parkinson's disease
Heather M. Klintworth
Department of Environmental and Occupational Health Sciences – Toxicology, University
of Washington, Seattle, WA.
Parkinson’s disease results in the loss of dopaminergic neurons and leads to a decrease of
dopamine production in the brain. This leads to bradykinasia, loss of postural reflex and
tremors at rest. Currently, the causes and mechanisms underlying Parkinson’s disease are
still unknown. Epidemiological studies have shown an association between pesticide use,
in particular paraquat, and Parkinson’s disease. Some experiments have shown that
paraquat can induce dopaminergic cell death in animal models. By using PC12 cell lines,
we can utilize molecular biology tools and assays to determine if the JNK and p-38
signaling pathways are involved in paraquat exposure and possibly Parkinson’s disease.
Such studies could prove useful in developing new treatments for Parkinson’s disease by
elucidating new drug targets and increasing the awareness for the consequences of
pesticide exposure.
Ethanol and acetaldehyde-mediated developmental toxicity and apopotitic cell death
in zebrafish
Jane K. La Du, Mark J. Reimers, Amanda R. Flockton, and Robert L. Tanguay.
Department of Environmental and Molecular Toxicology. Oregon State University,
Corvallis, OR.
Ethanol is a well-established developmental toxicant. However, the mechanism(s) of this
toxicity remains unclear. Zebrafish are becoming an important model system for the
evaluation of chemical and drug toxicity. In this study, zebrafish embryos were utilized to
compare the developmental toxicity resulting from either ethanol or acetaldehyde
exposure. Embryos were exposed to waterborne ethanol and acetaldehyde concentrations
for various lengths of time (up to 48 hours post-fertilization, hpf) that encompass the
earliest stages of embryogenesis. The waterborne ethanol concentration that causes 50%
mortality (LC50) following a 45-hour ethanol exposure was approximately 340 mM (1.98%
v/v). When the exposure period was reduced to the first 24 hours of embryogenesis, similar
signs of toxicity were produced at nearly identical ethanol concentrations. Embryos
exposed to waterborne acetaldehyde that resulted in similar, but not identical, endpoints as
those induced by ethanol. Embryos were, however, almost three orders of magnitude more
sensitive to acetaldehyde than to ethanol. These reproducible endpoints for ethanol and
acetaldehyde included pericardial edema, yolk sac edema, axial malformations, otolith
defects, delayed development and axial blistering. Ethanol and acetaldehyde both
negatively impacted embryonic development, however ethanol was more teratogenic based
on Teratogenic Indices (TI). Since ethanol was developmentally toxic to embryos attention
is now on the potential mechansim(s) of action. Ethanol associated craniofacial
abnormalities have been observed in mammals and in this study. The occurrence of these
malformations has been suggested to be partly due to apoptotic cell death of neural crest
cells that are critical for craniofacial morphology. Using assays to measure apoptosis, there
was a marked increase in apoptotic cells in the brain region of ethanol exposed embryos.
These results demonstrate that the zebrafish model will provide an opportunity to further
evaluate the mechanism of action(s) of ethanol on vertebrate development. (Supported by
NIH grants AA12783, ES03850, and ES00210)
Generation and characterization of a glutamate-cysteine ligase modifier subunit null
mouse
Lisa A. McConnachie, Francesca N. Hudson, Carol B. Ware, Carolina Fernandez, Portia
A. Vliet, Colin C. White and Terrance J. Kavanagh
Departments of Environmental and Occupational Health Sciences, Pathology, and
Comparative Medicine, University of Washington, Seattle, WA.
The rate-limiting step in glutathione (GSH) biosynthesis is catalyzed by glutamate cysteine
ligase (GCL). This enzyme is a heterodimer composed of a catalytic and a modifier
subunit. The modifier subunit, GCLM, functions to modify the activity of GCL by
increasing its affinity for glutamate and attenuating the feedback inhibition of GCL by
GSH. To more thoroughly characterize the role of GCLM in GSH biosynthesis, we have
created a GCLM-null mouse model. We replaced exon 1 of the GCLM gene with a galactosidase/neomycin phosphotransferase fusion protein, allowing in situ detection of
GCLM promoter activity. Western blots performed on tissues, including liver and kidney,
indicate the -/- (null) mice do not express immunodetectable levels of GCLM while the +/mice express GCLM at 50% of wild-type (+/+) controls. Furthermore, hepatic GCL
activities in -/- and +/- mice are 23  9% and 71  9% of controls while GSH levels were
22  6% and 96  9% of controls, respectively. Conversely, -galactosidase activity was
increased in the -/- and +/- mice when compared to the controls. We have generated
mouse embryo fibroblasts from these mice to serve as a complementary in vitro system to
evaluate GCLM-promoter driven induction. Treatment of these cells with the GCL
inducer tBHQ resulted in significant increases in -galactosidase activity. Treatment of
these same cells with ambient particulate matter also resulted in similar GCLM induction.
In vivo exposure of the GCLM null mice to diesel exhaust again yielded similar results.
These mice will be very useful in further characterizing the function of GCLM in oxidative
stress and GSH biosynthesis. (Supported by NIH grants 1P42ES04696, 1R01ES10849,
1T32ES07032, 1P30ES07033, and 5T32AG000057).
Mutagenic potential of an acrolein-derived DNA adduct,
γ-hydroxypropanodeoxyguanosine, in mammalian cells
Irina G. Minko1,2, Manorama Kanuri2, M. Todd Washington2, Satya Prakash2, Louise
Prakash2, Thomas M. Harris3, and R. Stephen Lloyd1,2
1
Center for Research on Occupational and Environmental Toxicology, Oregon Health and
Science University, Portland, OR, 2Sealy Center for Molecular Science, University of
Texas Medical Branch, Galveston, TX, 3Center in Molecular Toxicology, Vanderbilt
University, Nashville, TN
Acrolein, an α, β-unsaturated aldehyde, is a mutagenic ubiquitous environmental pollutant
produced from incomplete combustion of organic materials and is formed endogenously by
lipid peroxidation. The major acrolein-derived DNA adduct, γhydroxypropanodeoxyguanosine (γ-HOPdG), has been detected in mammalian tissues and
may play an important role in mutagenesis and carcinogenesis. While γ-HOPdG exists
primarily in its ring-closed form in single-stranded DNA, in duplex DNA, the cyclic
structure opens and the modified deoxyguanosine forms a normal Watson-Crick base pair
with a complementary cytosine. The mutagenic potentials were determined for γ-HOPdG
and structurally similar model adducts that mimic either the ring-closed or ring-opened
form of γ-HOPdG. In mammalian cells, γ-HOPdG and the ring closed analog yielded
comparable mutation frequencies (7.4 % and 8.3 %, respectively), while the ring opened
analog was essentially non-mutagenic. Replication assays were performed in vitro using a
variety of eukaryotic replicative and translesion DNA polymerases, including pol , pol ,
pol η, pol ι, pol κ, and Rev1/polζ. Based on both the mutational data and on the extensive
studies investigating the capability of each individual polymerase to perform DNA
synthesis past γ-HOPdG, we speculate that during replication, the mutagenic potential of
this adduct depends on at least two factors: (1) whether the adduct enters the replicative
fork in its ring closed or ring opened form, and (2) the efficiency of polymerase switch
mechanisms to recruit bypass DNA polymerases, proficient in synthesis past the adduct, to
a blocked replicative fork.
Cloning, expression, and function of Plasmodium falciparum glutamate cysteine
ligase: A potential target for antimalarial chemotherapy
Isaac Mohar and Terrance J. Kavanagh
Department of Environmental and Occupational Health Sciences, University of
Washington, Seattle, WA.
The need to identify and develop effective treatments for malaria is evident in the rapid
evolution of drug-resistant epidemics throughout the world. The nonspecific, and often
unknown, mechanism of the majority of antimalarials in combination with sub-lethal
dosing regimens may permit the further development of resistant parasites. The principal
aim of this study is to identify an inhibitor of the glutamate cysteine ligase (GCL) enzyme
of Plasmodium falciparum 3D7. Inhibition of P. falciparum GCL is hypothesized to
disrupt its ability to maintain adequate glutathione (GSH) levels and thereby disrupt the
redox status of infected red blood cells, suppressing the asexual reproduction phase of the
parasite lifecycle. Following successful cloning, expression, and purification of the P.
falciparum GCL, structural and functional comparisons will be conducted using Mus
musculus GCL, the mammalian homologue of P. falciparum GCL, as the mammalian
control. Screening of enzymatic inhibitors will be carried out beginning with a known
mammalian GCL inhibitor, buthionine sulfoximine, and molecules of similar structure.
Screening and identification of inhibitors specific for P. falciparum GCL will not only
provide a potentially highly efficacious (and possibly inexpensive) antimalarial
chemotherapy but also aid in the determination of the mechanism and regulation of
mammalian GCL as well as the potential of targeted disruption of GSH homeostasis as a
means of combating other parasitic infections.
(Supported by Puget Sound Partners for Global Health and NIEHS ES07032 EP/T
Training Grant)
In-vitro evidence of testicular stathmin reactivity to 1,2-diacetylbenzene
Sabri, M.I., Hashemi S.B., Chohan S., Khalil S., Tshala-Katumbay, D.D., Palmer, V.,
Cranson, A. Pounds, J.G. and Spencer P.S.
Center for Research on Occupational and Environmental Toxicology, Oregon Health &
Science University, Portland OR
Protein-reactive 1,2-diacetylbenzene (1,2-DAB) is the putative active gamma-diketone
metabolite of 1,2-diethylbenzene (1,2-DEB), a minor neurotoxic component of aromatic
solvents including jet fuels. Rats treated with 1,2-DEB or 1,2-DAB develop blue
discoloration of brain, greenish urine, altered brain stem auditory evoked potentials and
sensorimotor axonal neuropathy. Proteomic studies have focused attention on 1,2-DABinduced changes in stathmin, a cytosolic protein that binds to tubulin, regulates
microtubule assembly/disassembly, and is required for axonal integrity. Rats treated with
1,2-DAB, but not 1,3-DAB, also show prominent discoloration of testes and seminal
vesicles suggesting the neurotoxic isomer also targets these organs. Testes slices were
incubated with 1, 2, 5, or 10 mM 1,2-DAB or 1,3-DAB, or with the vehicle, for 30 min at
37 0C. Tissues were homogenized in sample buffer and 6-12 µg proteins separated by
SDS-PAGE. Electrophoresed proteins were transfered electronically to PVDF membranes,
probed with anti-stathmin antibody, and stathmin visualized colorimetrically by applying
secondary antibody conjugated with horse radish peroxidase (HRP) and streptavidin-HRP
amplifying reagent or fluorometrically by using a secondary antibody tagged with IR Dye800 and Odyssey Infrared Imaging System. 1,2-DAB reduced the intensity of the native
stathmin band in a concentration-dependent manner. The loss of stathmin was ~ 15% at 1
mM, ~ 20% at 2 mM, ~ 35% at 5 mM, and ~ 50% at 10 mM 1,2-DAB, with corresponding
increased adduct formation in the high molecular weight region. By contrast, 1-10 mM
1,3-DAB under similar conditions had no effect on stathmin or adduct formation. Rat
brain slices treated with 1-10 mM 1,2-DAB, but not 1,3-DAB, showed a similar, but not
identical, pattern of stathmin loss. Taken together, the results of this study suggest 1,2DAB (like its aliphatic gamma diketone cousin 2,5-hexanedione) is not only a neurotoxin
but also targets testes. [Supported by NIEHS grants ES10338 and ES 11384 and Oregon
Workers' Benefit Fund].
The mechanisms of dithiocarbamate and isothiocyanate developmental toxicity in the
zebrafish early development model
F. Tilton, M.Vue and Robert L. Tanguay
Department of Environmental and Molecular Toxicology and Marine Freshwater
Biomedical Sciences Center, Oregon State University, Corvallis, OR.
The dithiocarbamate pesticide, sodium metam (NaM), and its active ingredient,
methylisothiocyanate (MITC), cause notochord malformations in the developing zebrafish
during a sensitive developmental window which occurs > 6 hours before the visual
manifestation of the malformation. In this study, several isothiocyanates and
dithiocarbamates were investigated in the zebrafish model to determine the structural and
teratogenic relationships that may aid in elucidating the mechanism(s) of notochord
malformation. Isothiocyanates (ITC) and dithiocarbamates (DTC) ranging from natural
and synthetic pesticides, cancer chemoprevention agents, pharmaceuticals, and
environmental degradation products were exposed to the developing embryo from 4 to 24
hours post fertilization (hpf). Among other observations, all the dithiocarbamates elicited
notochord malformations similar to those previously observed with NaM and MITC. The
DTC LC50s ranged from 40 ppb to 4 ppm, EC50s from 4.0 to 400 ppb and NOELs from
<4.0 to 40 ppb. Of the ITC tested, only MITC exposure led to notochord malformations.
The potential involvement of cellular oxidative stress and metals is currently under
investigation. Reduced glutathione (GSH) suppressed DTC-induced notochord
malformation with toxicant co-exposures; however, differences in GSH
protection/recovery were evident when the exposure windows were manipulated.
Surprisingly, GSH failed to diminish MITC-induced malformations. Preliminary studies
using Cu, Zn, Fe as well as their respective membrane permeable and impermeable
chelators, indicate a potential contribution from Cu. In conclusion, the mechanism of
developmental notochord malformation from DTC exposure appears to require in vivo
glutathione depletion prior to DTC-target interaction and metals may play some role in the
mechanism leading to notochord malformation. (Supported by NIEHS #ES00210,
#ES03850, and #ES07060)
Evidence of aflatoxin b1 hepatic tumor promotion by the dietary constituent
3,3’-diindolylmethane: tumor incidence and gene expression profiles
S. C. Tilton1,2,4, J. D. Hendricks1,4, G. S. Bailey1,2,3,4 and D. E. Williams1,2,4
1
Department of Environmental and Molecular Toxicology, 2The Linus Pauling Institute,
The Environmental Health Sciences Center and 4The Marine and Freshwater Biomedical
Sciences Center, Oregon State University, Corvallis, OR.
3
Dietary indoles, indole-3-carbinol (I3C) and 3,3’-diindolylmethane (DIM), from
cruciferous vegetables are available as over-the-counter supplements and are currently
being evaluated in clinical trials as potential chemopreventive agents against breast and
ovarian cancers. However, I3C has also been found to act as a tumor promoter in rat
(multi-organ) and trout (liver) models when fed long-term post-initiation and evidence
suggests that estrogenic mechanisms might be important. DIM is the major acid
condensation product of I3C in the stomach after dietary consumption and in the liver after
absorption and was previously found to be estrogenic in trout liver based on global gene
expression profiles and receptor binding assays. In this study, we demonstrate that when
given post-initiation, dietary DIM promotes aflatoxin B1 (AFB1)-induced
hepatocarcinogenesis in the rainbow trout model similar to 17-estradiol (E2), a known
hepatic tumor promoter. Trout embryos were initiated with 50 ppb AFB1 and then 3month old fish were fed diets containing 120 and 400 ppm DIM or 5 ppm E2 for 18 weeks.
Fish were sampled for liver tumors at 10 months. Both tumor incidence and multiplicity
were elevated in AFB1-initiated trout fed 400 ppm DIM and 5 ppm E2. To evaluate the
mechanism of tumor promotion, gene expression profiles have been examined in liver
tumors and normal adjacent tissue of AFB1-initiated animals and those also exposed to
dietary tumor promoters. Preliminary results from the rainbow trout 70-mer
oligonucleotide array suggest that distinct genes are regulated in tumors compared to
adjacent normal tissue and also in tumors from promoted animals compared to initiatedonly. These findings may lead to better understanding of the mechanism of hepatic tumor
modulation by dietary indoles. Supported by NIH grants ES03850 and ES07060.
Structural requirements for hydrocarbon axonopathy
Tshala-Katumbay, D.D.1, Palmer, V.S.1, Dixon, D.A.2, Deinzer, M.L.3, Sabri, M.I.1,
Spencer, P.S.1
1
Oregon Health & Science University, CROET, Portland, OR, 2University of Alabama,
Dept. of Chemistry, Tuscaloosa, AL, 3Oregon State University, Dept. of Chemistry,
Corvallis, OR
Rodents receiving the protein-reactive aromatic gamma-diketone 1,2-diacetylbenzene (1,2DAB), but not non-reactive 1,3-DAB, develop blue tissue coloration and proximal CNSPNS neurofilamentous (NF) axonopathy. The chromogen is an isoindole product of
gamma-diketone reaction with epsilon-amino groups of lysine, including those in NF
proteins. We asked whether 1,2,4-triethylbenzene (1,2,4-TEB) shows comparable
structure-dependent chromogenic and neurotoxic properties. Male 12-week-old C57 BL/6
mice were dosed by gavage every other day, 3 days/week, with 300-1000 mg/kg 1,2,4TEB, 1,3,5-TEB or vehicle (olive oil) for up to 10 weeks. Mice treated with 1,2,4-TEB, but
not 1,3,5-TEB or vehicle, excreted greenish urine and developed hindlimb
weakness. Blue-green colored nervous tissue showed medullary, spinal and root axonal
swellings from presumed proximal block of NF transport. Mice treated with 1,3,5-TEB or
vehicle lacked tissue coloration, hindlimb weakness and neuropathology. These results
show 1,2,4-TEB, the parent of 1,2,4-TAB (with the 1,2-diacetyl moiety required for
protein reactivity), but not 1,3,5-TEB (without the ortho-diethyl structure), is chromogenic
and neurotoxic. This suggests the presence of 1,2-spaced ethyl moieties on a benzene ring
is required for chromogenic and neurotoxic properties of aromatic hydrocarbons. [NIEHS
grants ES10388 & ES11384]
Effect of ethanol on NGF and BDNF secretion in rat astrocytes
Kathryn VanDeMark, Marina Guizzetti, and Lucio G. Costa
Department of Environmental and Occupational Health Sciences, University of
Washington, Seattle, WA
Fetal Alcohol Syndrome is a widespread problem resulting in several lifelong, physically,
and mentally disabling conditions. It is one of the leading known preventable causes of
mental retardation and birth defects. While most of the research directed at identifying the
mechanisms of the developmental neurotoxicity of ethanol has focused on the effects on
neurons, new findings suggest that glial cells might be a primary target of ethanol toxicity
and that alterations in neuron-glial interactions can lead to neuronal abnormalities and
participate in ethanol-induced teratogenesis. Astrocytes make up the largest percentage of
cells in the central nervous system and provide both mechanical and metabolic support for
neurons. One means of providing this support is by secreting neurotrophic factors. These
factors are essential for neuronal cell differentiation, growth, and survival, and they help
mediate fetal development. The principal aim of this study is to determine the effect of
ethanol on astrocyte secretion of two particular neurotrophic factors, nerve growth factor
(NGF) and brain-derived neurotrophic factor (BDNF). Protein secretions are currently
measured using enzyme-linked immunosorbant assay (ELISA) systems and mRNA levels
will be examined by RT-PCR. Following these results, the hypothesis that some of the
effects of ethanol on neurons may be due to inhibition of the release of neurotrophic
factors from actrocytes will be investigated. This research will provide new insights on the
regulation of neurotrophic factor release from astrocytes during brain development, on the
mechanisms underlying the developmental neurotoxicity of ethanol, and on how
environmental toxicants may indirectly affect neuronal functions and survival by exerting
an inhibitory effect on glial cells.
(Supported by grants AA-08154 and NIEHS ES07032 EP/T Training Grant)
Videotape Presentations
Courtesy of Third World Medical Research Foundation
Sunday at 9:00 a.m. (Platform)
Nodding Disease, TWMRF, Visual images of an acquired convulsive disorder commonly
associated with onchocerciasis and triggered by the presentation and eating of local food.
Location: Southern Sudan. Presented by Peter Spencer.
Sunday at 9:10 a.m. (Platform) and Poster Session (cycled video)
Seychelles Paraplegia, TWMRF (~15 min). Documentary of a progressive HTLV-1associated neurological disease that was originally attributed to fish-derived mercury
toxicity. Location: Mahe, Seychelles (Indian Ocean Island of Africa). Mentioned by
Valerie Palmer.
Sunday at 9:10 a.m. (Platform)
Lathyrus and lathyrism, TWMRF. Visual images of lathyrism, a form of spastic
paraparesis caused by food dependency on grasspea (Lathyrus sativus). Presented by
Valerie Palmer
Sunday at 9:40 a.m. (Platform) and Poster Session (cycled video)
Konzo, Uppsala (~15 Min). Documentation of spastic paraparesis associated with cassava
food dependency in sub-Saharan Africa. Location: Democratic Republic of Congo.
(Kindly provided by Thorkild Tylleskar, M.D.) Presented by D. Desire Tshala-Katumbay.
Sunday at 10:20 a.m. (Platform) and Poster Session (cycled video)
The Poison that Waits? BBC London [1988] (~60 min.). Documentary of research on
cycad-associated western Pacific amyotrophic lateral sclerosis and parkinsonism-dementia
complex and its relationship to look alike disorders worldwide. Location: Guam, Japan,
New Guinea, UK and USA. Presented by Peter Spencer.