21ST ANNUAL MEETINGPROGRAM SCHEDULE AND ABSTRACTS

2004 Theme:

Toxicology in

Third World Settings

MT. BACHELOR VILLAGE RESORT CONFERENCE CENTERBEND, 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 least-developed 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 SpencerPANWAT President2004-2005

Program and Meeting Schedule (Conference Center)

Friday, September 17 th :

6:00 to 9:00 pm Meeting registration (Spring Village Canyon Foyer East)6:00 to 9:00 pm Submit platform presentations to AV specialist Dan Austin (Winter’s Hope B)6:00 to 9:00 pm PANWAT Officers meeting, Café Rosemary

1110 NW Newport AvenueBend, OregonPh: 541-317-0276

Saturday, September 18 th :

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)

7:00 to 8:25 am Poster set up (Winter’s Hope B)7:00 to 8:25 am Meeting registration (Foyer) 7:45 to 8:30 am Breakfast (Winter’s Hope A)

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 18 th :

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 19 th :

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 Parkinsonism-Dementia 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 ContributionInvited speakers with John Tor Agbidye and Richard Pleus

2:00 to 2:15 pm Graduate Student Award PresentationsPlatform 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=22. Ski House Room (one bedroom), one queen in bedroom and one pull out couch in living room, $893. 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 theirown 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: 503-494-2517) 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

PresidentPeter SpencerOHSU, Center for Research on Occup. & Env. Tox. (CROET)3181 SW Sam Jackson Park RoadCROET L606Portland, OR 97239Ph: (503) 494-4273Fx: (503) 494-4278spencer@ohsu.edu

Vice PresidentRosita Rodriguez - ProteauOregon State UniversityPharmaceutical Sciences203 Pharmacy BuildingCorvallis, OR 97331Ph: (541) 737-5786Fx: (541) 737-3999rosita.rodriguez@orst.edu

Vice President-ElectAndrij HolianUniversity of MontanaCenter For Env. Health Sciences153 Skaggs Building32 Campus DriveMissoula, MT 59812Ph: (406) 243-4018Fx: (406) 243-2807aholian@selway.umt.edu

Secretary/TreasurerCarin ThomasCentral Washington UniversityDept of ChemistryMS 7539Ellensburg, WA 98926-7539Ph: (509) 963-2815Fx: (509) 963-1050cthomas@cwu.edu

Councilor: (2 year term)Cecile KrejsaPre-clinical DevelopmentZymoGenetics, Inc.1201 Eastlake Ave. ESeattle, WA 98102(206) 515-4974krejsac.zgi.com

Councilor: (1 year term)David M. Shepherd, Ph.D.Center for Env. Health SciencesSkaggs Building, Room 5832 Campus DriveUniversity of MontanaMissoula, MT 59812Ph: (406) 243-2224 Fx: (406) 243-2807 david.shepherd@umontana.edu

Past PresidentMarc W. Fariss, Ph.D.College of PharmacyWashington State UniversityP.O. Box 656534Pullman, WA 99164-6534Ph: (509) 335-4689fariss@mail.wsu.edu

Student Representative Castle FunatakeOregon State UniversityEnvironmental and Molecular Toxicology1007 Agricultural Life SciencesCorvallis, OR 97331Ph: (541) 737-2096Fx: (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 UnitInternational 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 cis-urocanic 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 trans-urocanic 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 post-infection, 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 KodakCompany, 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 0.5-0.8 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 individualsexposed 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-1-associated 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-N-oxalylamino-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. Low-toxin 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 (2-hydroxyisobutyronitrile-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 2-iminothiazolidine-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 Parkinsonism-Dementia 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 environmentsthrough 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 aberrantcrypt foci: Evidence for differential processing of PhIP adduct mispairs by themismatch 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 DNAcontaining site-specific butadiene-derived N3-RS uracil adducts

Priscilla H. Fernandes1, Richard P. Hodge2, and R. Stephen Lloyd1

1Center 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 indendritic 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) peptide-specific 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 TCDD-treated 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

1Department 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 S-hydroxymethlyglutathione 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

1Department 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 #R-827355-01-0 and NIEHS #P30 ES07033.)

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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

1College 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 restricted-use 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

1College 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 thedeveloping 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

1NSF-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 anti-inflammatory 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 dose-dependent 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

1CROET, 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 isdistinct when compared with other DNA damaging agents

G.E. Kisby1; M. Standley2*; P. Pattee2; J. O’Malley2; J. Muniz1; A. Olivas1; S.R. Nagalla2

1CROET, 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 sub-lethal 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

1Oregon State University (OSU), Corvallis, OR 97330; 2Gonzaga UniversitySpokane, 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 1,2-DAB 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 deathin 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 nullmouse

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

1Center 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, γ-hydroxypropanodeoxyguanosine (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-DAB-induced 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 Dye-800 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 1,2-DAB (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

1Department of Environmental and Molecular Toxicology, 2The Linus Pauling Institute, 3The Environmental Health Sciences Center and 4The Marine and Freshwater Biomedical Sciences Center, Oregon State University, Corvallis, OR.

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 3-month 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 initiated-only. 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 (1,2-DAB), but not non-reactive 1,3-DAB, develop blue tissue coloration and proximal CNS-PNS 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 1,2,4-TEB, 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 PresentationsCourtesy 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-1-associated 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.