[] Intrntnl Neurotoxicology Assoc 1994 Cognitive Dysfunction Environmental Exposure (fwd)

[Guest guest]

iequality

Subject: [iequality] Intrntnl Neurotoxicology Assoc 1994 Cognitive Dysfunction

Environmental Exposure

_International Neurotoxicology Association INA 5 Abstracts_

(http://www.neurotoxicology.org/ina5_abstracts.htm) 1994

Wow! Look where we were headed before science got Bushwacked.

Sharon

Abstracts of the 5th Meeting of the International Neurotoxicology Association

THE HUMAN GENOME PROJECT: NEW TECHNOLOGIES FOR DNA EXPLORATION, OR LOOKING

FOR THE GEORGE BURNS GENOTYPE

Deborah A. Nickerson

Department of Molecular Biotechnology, University of Washington, Seattle, WA

98195

One of the most challenging problems in the study of biology and medicine is

related to defining the connection between phenotype, the observable

characteristics of an individual, and genotype, the DNA sequence of the

individual.

Over the next decade, the Human Genome Project will provide a composite

sequence of the 24 different human chromosomes and will uncover the information

stored in these structures, i.e. the 100,000 or so genes responsible for human

structure, function, growth and development. The Human Genome Project is

unique, based not only on its size and scope but also based on its dependence

on

the development of new technologies. Current and newly emerging technologies

from this project will be presented and discussed in regard to their role in

the next generation of medicine and its implications in understanding the

diversity of the human phenotype.

THE LATENT ROOTS OF NEURODEGENERATIVE DISEASE

Bernard Weiss

Department of Environmental Medicine, University of Rochester School of

Medicine and Dentistry, Rochester, NY

Satchel Paige, along with his extraordinary athletic accomplishments, is

also remembered as a philosopher. Although no one ever thought of him as an

expert on neurodegenerative disease, he was perspicacious enough to enunciate a

useful guiding principle: " Don't look back; something may be gaining on you. "

That something is always gaining on us is certain, and we all try to elude

it, but looking back is what scientists, in our arrogance, do. We all accept

the Paige principle that neurodegenerative diseases begin long before they

erupt into perceptible disability. We cannot always state whether the process

is

inherently abnormal, or an acceleration of natural, universal aging, or a

confluence of the two. The puzzle of how the process advances is one of the

most

profound challenges to neuroscience, but many clues tell us that aging must

be accorded at least a secondary role: 1. Neurobehavioral function declines

with age. 2. The incidence of neurodegenerative disease shows a clear

age-related acceleration. 3. Key areas of the brain lose neurons with age,

restricting their ability to compensate for damage. 4. Apparent recovery from

an

earlier neurological disability may revert with age. Any research agenda aimed

at

the etiology of neurodegenerative disease must deal with both onset and

process. For investigators, it means no acceptable substitute for meticulous

longitudinal studies in both the clinic and the laboratory, carried out with

the

most sensitive instruments available.

ORGANOPHOSPHATE-INDUCED DELAYED NEUROTOXICITY (OPIDN): FACTS AND FANTASIES

S. Padilla 1 P. Tandonand 2, M. Ehrich 3 and C.N. Pope 4 1Neurotoxicity

Division, U.S. Environmental Protection Agency, Research Triangle Park, NC

2 Harvard University, Boston, MA

3 Virginia Tech. University, Blacksburg, VA

4 Toxicology Program, School of Pharm., N. L.U., Monroe, LA

OPIDN is one of the few neuropathic conditions where the association between

exposure to a neurotoxic compound and the development of neurodegeneration

is well-recognized. Exposure to a certain subset of organophosphate

anticholinesterase compounds (some are environmentally relevant) produces a

central-peripheral distal axonopathy in man and many experimental animals. This

axonopathy does not occur immediately after exposure; the development of the

clinical

signs is usually delayed approximately 14 to 21 days after an acute

exposure. Although over 60 years has elapsed since the initial identification

of

OPIDN, there has yet to be an elucidation of the mechanism of this neuropathy.

It

is known that the neuropathy can be prevented or potentiated by other

esterase inhibiting compounds (generally depending on the relative sequence of

administration of both compounds) and that young animals are particularly

insensitive to the development of the neuropathy. OPIDN will be discussed in

the

light of these developments as well as more recent data and hypotheses

regarding

the balance of neuronal degeneration and regeneration normally maintained by

growth/trophic factors.

EVIDENCE FOR DELAYED METHYLMERCURY NEUROTOXICITY IN MONKEYS

D. C. Rice

Toxicology Research Division, Department of Health, Ottawa, Ontario

As a consequence of the tragic outbreaks of human organic mercury poisoning

in Japan and Iraq, substantial research effort has focused on characterizing

the developmental effects of exposure to methylmercury in animal models,

including studies in macaque monkeys (Macaca fasicularis) at the Canadian

Health

Protection Branch. One group of five monkeys, presently 19 years old, was

dosed with 50 µg/kg/day of mercury as methylmercuric chloride from birth to 7

years of age; blood mercury concentrations during the period of dosing were

approximately 0.75ppm. In another study examining the effects of in utero plus

postnatal exposure in the same species of monkey, females were dosed with 10,

25, or 50 µg/kg/day of mercury as methylmercuric chloride; blood mercury

levels averaged 0.37, 0.75, or 1.42 ppm during pregnancy. One, two, and five

infants were born from the three dose groups, respectively. One infant in the

high-dose group was born with signs of methylmercury poisoning resembling those

of human infants, including motor impairment and nystagmus. When the group of

monkey exposed only postnatally until 7 years of age was 13 years old,

individuals began exhibiting clumsiness not present previously. Further

exploration revealed that treated monkeys required more time to retrieve treats

than

did non exposed monkeys and displayed abnormalities on a clinical assessment of

sense of touch in hands and feet, despite the fact that clinical

examinations performed routinely during the period of dosing had not yielded

abnormal

results. This observation was pursued in both groups of monkeys by objective

assessment of somatosensory function in the hands: both groups of monkeys

exhibited impaired vibration sensitivity. These results are strongly suggestive

of

a delayed neurotoxicity manifested when these monkeys reached middle age.

These data are consistent with data in mice exposed developmentally to

methylmercury (J.M. Spyker, Fed. Proc. 34, 1835, 1975), in which abnormalities

including kyphosis, obesity, and severe neurological deficits were observed

only as

the animals aged.

NEUROTOXINS AND NEURODEGENERATIVE DISEASE

J. Langston

During the last decade there has been increasing interest in the

relationship between neurotoxins and neurodegenerative disease. There are

several

reasons for this. First, a number of toxins have been discovered which are

remarkably selective for the same neural populations that are affected in human

neurodegenerative diseases, including Parkinson's disease and Huntington's

chorea.

Such compounds can be used to model these diseases, an approach that has

wide-ranging experimental and clinical implications. Secondly, there is the

important possibility that investigating the mechanism of action of these

compounds may provide insights into the mechanisms underlying the actual

process of

neuronal degeneration. Finally, in at least some instances, there is the

provocative possibility that similar compounds in the environment might even be

a

cause of the disease. In this presentation, 1-methyl-4-phenyl- 1,2,3,6-

tetrahydropyridine or MPTP will be discussed as the prototype of such a toxin.

This compound meets all of the criteria for what might be called a

" neurodegenerative neurotoxin " : it induces degeneration of specific neuronal

populations

after systemic administration which are nearly identical to those which are

affected in a neurodegenerative disease (in this case Parkinson's disease).

The discovery of the biological effects of this compound, which induces

degeneration of dopaminergic neurons of the substantia nigra, and a

corresponding

profound dopamine depletion in the striatum of human and non-human primates,

has already lead to the first good animal model for the disease. But there are

many other lessons that have been learned as a results of studies on the

mechanism of action of MPTP as well. For example, it is now weel known that

MPTP

is biotransformed to its toxic metabolite, 1-methyl-4-phenypyridinium ion,

via MAO B. This process appears to take place in astrocytes. It could be argued

that this a precedent-setting phenomenon, in that it may be the first

example of the brain's own enzymatic machinery generating a selective

neurotoxin

from a non-toxic compound. Since MAO B can be easily blocked pharmacologically,

this observation has already lead to the development of a neuroprotective

strategy for Parkinson's disease. Other aspects of MPTP neurotoxicity, such as

its effects on Complex I of the mitochondrial respiratory chain, have greatly

enhanced interest in the role of energy metabolism and neurodegenerative

disease. These and other features of MPTP neurotoxicity, and its broader

implications for neurotoxins and neurodegenerative disease, will be reviewed,

along

with implications for future research in this interesting and rapidly

developing field.

POLYCHLORINATED BIPHENYLS DECREASE DOPAMINE AND ELEVATE GABA CONTENT IN

STRIATAL SLICES FROM ADULT RATS

R.F. Seegal, M.A. Chishti and G. Battaglioli

Wadsworth Center, New York State Department of Health, Albany, NY

Polychlorinated biphenyls (PCBs) are widespread environmental neurotoxicants

associated with behavioral changes in human infants, behavioral and

neurochemical changes in experimental animals and reductions in dopamine (DA)

content

of pheochromocytoma (PC12) cells in vitro. To study the neurochemical

effects of PCBs in a more complex ex-vivo system, we exposed 350-µm thick

striatal

slices from adult rats to varying concentrations of a 1:1 mixture of Aroclors

1254 and 1260 and measured slice content of DA and gamma-aminobutyric acid

(GABA) by HPLC. PCBs reduced slice DA content in a time and dose dependent

manner with a 65-80% reduction observed after 6 h at 200 ppm. Accompanying

these

reductions in slice DA content were significant dose-related increases in

slice GABA content. Maximal elevations in slice GABA content (a doubling

compared to vehicle-exposed slices) were observed at PCB concentrations in

media

equal to or greater than 100 ppm. GABA and DA in the basal ganglia are mutually

inhibitory, complicating interpretation of the PCB-induced alterations in

these neurotransmitters. Reductions in in-vivo striatal DA content by

neuroleptics or 6-OH DA elevate GABA content. However, in preliminary studies,

a-methyl-_-tyrosine (an inhibitor of DA synthesis) reduced slice DA content by

50%

but did not alter slice GABA content. This result suggests that the elevations

in GABA are a direct consequence of PCB exposure and not secondary to

PCB-induced reductions in slice DA content. A cause and effect relationship, if

any, between changes in GABA content and changes in DA content remains to be

investigated. Supported by NIEHS Grant #ES04913.

DEVELOPMENTAL EXPOSURE TO AROCLOR 1254 CAUSES HYPOTHYROIDISM AND

LOW-FREQUENCY HEARING LOSS IN RATS: ATTENUATION OF EFFECTS BY THYROXIN

REPLACEMENT

E. S. Goldey, L. S. Kehn & K. M. Crofton

Heightened interest has focused on the possible role of endocrine disruption

in mediating the effects of developmental neurotoxicants. The auditory

system is dependent upon thyroid hormones for normal development, and we

reported

that developmental PCB exposure caused low-frequency hearing loss and

hypothyroidism in rats. These findings lead us to suggest that PCB-induced

disruption of this endocrine axis may have contributed to the observed auditory

deficits. To further investigate this possibility, primiparous Long- rats

received daily oral doses of corn oil (control) or 8 mg/kg of Aroclor 1254 from

gestation day (GD) 6 through postnatal day (PND) 21. In addition, from PND

4-21, all pups received daily, subcutaneous injections of saline or 100 µg/kg

thyroxin to yield four groups of litters: corn oil plus thyroxin (CO-T4), corn

oil plus saline (CO-S), Aroclor 1254 plus thyroxin (PCB-T4), and Aroclor 1254

plus saline (PCB-S). We measured thyroid hormone (T4 and T3) concentrations

in serum collected from pups on PND 7, 14 and 21. On PND 7 and 21, we also

monitored the kinetics of the hormones following T4 injection in the CO-T4 and

PCB-T4 groups at 1, 3, 5, 8 and 24 hours post injection. On PND 7, 14 and 21,

T4 levels were dramatically depleted in the PCB-S and PCB-T4 groups compared

to the CO-S group. The thyroid hormone levels in the CO-T4 pups remained

significantly elevated over all other groups on each sample day, although no

other physical or functional alterations were seen in this group compared to

CO-S. The kinetics studies revealed that thyroxin injection raised circulating

hormone levels in the PCB-T pups to near CO-S levels for approximately 6 hours

post-injection, and that levels fell precipitously thereafter. As in our

previous studies, reflex modification audiometry revealed low-frequency (1

kHz),

auditory threshold deficits in adult offspring of Aroclor 1254-exposed dams.

Importantly, the auditory effects of PCBs were significantly attenuated,

although not eliminated, by thyroxin replacement therapy. Further, the T3 and

T4

kinetics indicate that the incomplete attenuation of the auditory deficit in

the PCB-T4 group may be due to the relatively short half-life of the

injected T4. These findings strongly support our earlier suggestion of a

causative

link between PCB-induced hypothyroidism and disruption of cochlear development

and function.

PRENATAL EXPOSURE TO POLYCHLOROBIPHENYLS: PCB METABOLISM, THYROID HORMONE

HOMEOSTASIS AND BRAIN DEVELOPMENT IN THE RAT

Dennis C. Morse1 , Abraham Brouwer1, Kor J. van den Berg2 , F.

Seegal 3

1 Department of Toxicology, Agricultural University, Wageningen, The

Netherlands

2 ITV-TNO, Rijswijk, The Netherlands

3 New York State Department of Health, Albany New York

The underlying mechanisms of PCB-induced developmental neurotoxicity are

unclear, although

it is a plausible hypothesis that pre- or postnatal PCB exposure indirectly

affects brain development by transiently reducing the amount of thyroid

hormone in the brain. We therefore examined the effects of pre- and postnatal

PCB

exposure on thyroid hormone levels in the plasma and brain of developing

rats, the mechanisms involved in altered thyroid hormone homeostasis and which

brain regions and cell types were affected. The results indicate that when

pregnant rats are exposed to Aroclor 1254, there is a substantial accumulation

of

the hydroxylated PCB metabolite, 2,4,5,3',4'-pentachloro-4-biphenylol

(4-OH-pentaCB) in the fetal plasma and brain. The accumulation of 4-OH-pentaCB

in

the plasma is probably responsible for the dramatic reductions in plasma and

brain T4 concentrations by blocking the transport of thyroid hormone to the

fetus. However, the fetal brain may be able to compensate for the decreases in

T4, by increasing the conversion of T4 to T3. Despite the lack of an observed

effect of maternal PCB exposure on brain T3 levels in the offspring, the

levels of a neurotypic protein (synaptophysin) and a glial protein (glial

fibrillary acidic protein, GFAP) as well as serotonin metabolism were altered

in

the brain of adult offspring in a complex fashion. The most prominent

neurochemical alterations were found in the lateral olfactory tract, prefrontal

cortex

and the brainstem. The neurochemical data could be interpeted as the result

of a primary lesion in the brainstem early in development.

EARLY DEVELOPMENTAL PCB-EXPOSURE AND NEUROLOGICAL/ NEUROBEHAVIORAL OUTCOME:

AN INTERIM REPORT FROM THE GERMAN PERINATAL STUDY*

G. Winneke1, A. Bucholski1, U. Krämer1, B. Heinzow2, B. Seidel3, J.

Walkowiak1, S. Weipert3, A. Wiener4, E. Schmidt3, and H.J. Steingrüber4

1 Medical Institute of Environmental Hygiene, University of Düsseldorf

2 Laboratory of Environmental Toxicology of Schleswig-Holstein, Kiel

3 Pediatric Clinic, University of Düsseldorf

4 Institute of Medical Psychology, University of Düsseldorf

In order to clarify uncertainties regarding spectrum and persistence of

developmental PCB-effects, an European coordinated prospective study was

initiated with study cohorts from the Netherlands, Denmark and Germany. The

ongoing

German study is based on PCBs 138, 153 and 180 in cordblood serum and

maternal milk. Measures taken at ages 15-20 d, 7 and 18 mo include

neurodevelopmental status (TOUWEN/PRECHTL) BAYLEY II, the FAGAN-test, as well as

vocalization-analyses. In addition TSH-levels are measured in cordblood and at

age 5 d.

With PCBs ranging from 0.05-0.64 µg/g fat in serum preliminary analyses based

on 103 infant- and 45 7 mo data sets do not yet suggest significant

exposure/outcome associations, but are indicative of a borderline positive

association

between cordblood PCB and TSH-elevation, suggestive of subtle hypothyroid

dysfunction.

*Partly supported by the CEC (Brussels), contract No. EV5V-CT92-0207

METALLOTHIONEIN IN ASTROCYTES: WHAT POSSIBLE SIGNIFICANCE?

M. Aschner

Department of Physiology and Pharmacology, Bowman Gray School of Medicine,

Winston-Salem, NC

The biological functions of metals and their accumulation within cells are

invariably linked to the existence of specific metal-binding proteins. Novel

kinds of -SH based metal clusters that have recently come o attention are the

metallothioneins (MTs). MTs are 6-7 kD in MW, and they contain some 60 amino

acid residues, among them 20 cysteines. Recent in vivo and in vitro studies

suggest that methylmercury (MeHg) neurotoxicity may be associated with

astrocyte dysfunction, leading to their failure to adequately control the

extracellular microenvironment. To explore the possibility that astrocytic MTs

afford a

protective role in heavy metal-induced neurotoxicity, we investigated the

effects of MeHg on the induction of MTs, and the ability of cultures which

express elevated MT levels to protect against the cytotoxic effects of MeHg. MT

mRNA was detected in untreated cells, suggesting constitutive MT expression in

astrocytes. Expression of MT-I mRNA in astrocyte monolayers exposed to 2 x

10-6 M MeHg for 6 hours was increased approximately 2-fold over MT-I mRNA

levels in controls. Western-blot analysis revealed a time-dependent increase in

MTs. Consistent with the constitutive expression of MTs both at the mRNA level

and protein level, a time-dependent increase in MT-immunoreactivity was

noted in MeHg-treated cells. The cytotoxic effects of MeHg were measured by the

rate of astrocytic [3H]-D-aspartate uptake. Pre-exposure to CdCl2, a potent

inducer of MTs, completely reversed the inhibitory effect of MeHg on

[3H]-D-aspartate uptake which occurs in MeHg-treated astrocytes with

constitutive MT

levels. In summary, astrocytes constitutively express MTs; treatment with MeHg

increases MT expression, and increased MT levels attenuate MeHg-induced

toxicity. Increased MT expression may represent a generalized response to heavy

metal exposure, thus protecting astrocytes, and perhaps also indirectly,

juxtaposed neurons.

SCHWANN CELLS AS TARGETS OF NEUROTOXICANTS*

P. Morell and A.D. Toews

University of North Carolina, Chapel Hill, NC 27599

Schwann cells envelop axons of the peripheral nervous system. Many are

further specialized to produce and maintain myelin. Schwann cells communicate

with

neurons both during development (e.g., through a nerve growth factor and

receptor system) and even after maturation (ATP receptors on Schwann cells

presumably respond to neuronal activity). Schwann cells, and the process of

myelination, are preferentially vulnerable to neurotoxic insults during the

developmental period of maximal accumulation of myelin. Part of the

pathophysiology

of various disease states, and of certain neurotoxic insults, is a direct

effect on Schwann cells and/or the myelin they maintain, leading to primary

segmental demyelination. Dissection of the biochemical events involved is often

complicated by the fact that the metabolic insult involved also affects

neurons. An exception is the neuropathy caused by feeding tellurium to young

rats;

a highly synchronous primary demyelination results, but cessation of

tellurium exposure is followed closely by a period of rapid remyelination. The

lack

of detectable axonal degeneration allows examination of changes in gene

expression specific to the processes of demyelination and remyelination. We

have

elucidated the primary metabolic lesion involved (tellurium blocks cholesterol

biosynthesis) and many of the steps coupling this metabolic lesion to

demyelination. A useful byproduct of the study is characterization of a

sensitive

marker for any neuropathy resulting in even subtle alterations in the normal

relationship between the Schwann cell-myelin unit and the axon it ensheathes.

Upregulation of mRNA for the low affinity nerve growth factor receptor is a

sensitive marker of nerve damage which may be useful as a screen for

potentially neurotoxic compounds. *Supported by USHS grants ES-01104 and

NS-11615.

POTENTIAL ROLE OF GLIAL CELLS IN PARKINSON'S DISEASE

D. A. Di Monte

The Parkinson's Institute, Sunnyvale, CA

Although degeneration of dopaminergic cells of the nigrostriatal pathway

represents the main neuropathologic feature of Parkinson's disease, other

cells,

including glial cells, may play a role in this process. A clear example

supporting this concept arises from studies on the mechanism of toxicity of

1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). MPTP causes a parkinsonian

syndrome in humans which is clinically indistinguishable from idopathic

Parkinson's disease. In order to exert its toxic effects, however, MPTP needs

to be

metabolically activated to its fully oxidized pyridinium metabolite MPP+.

This conversion is catalyzed by monoamine oxidase (MAO) B and is likely to

occur

within glial cells. MPP+ is then released from glial cells and actively

accumulated into dopaminergic neurons (the ultimate target of MPTP/MPP+

neurotoxicity) via the catecholamine uptake system. It is noteworthy that glial

MAO B

activity increases with age, paralleling an age-dependent enhancement of

MPTP-induced neurotoxicity. The contribution of glial cells to nigrostriatal

degeneration may also result from their participation in the metabolism of

dopamine. Both MAO A and MAO B are present in glial cells ad catalyze the

oxidative

deamination of dopamine. This reaction generates H2O2 which in glial cells

could be scavenged by the glutathione/gluatathione peroxidase system. H2O2 may

also cross cell membranes, however, and cause greater damage to neuronal

cells because of their lack of defense mechanisms against oxygen radical

accumulation. A final potential route by which glial cells may play a role in

the

pathophysiology of Parkinson's disease is related to the age-dependent

intraglial accumulation of iron. Iron could act as a catalyst for the

bioactivation

of MPTP-like neurotoxicants as well as for the conversion of dopamine into

reactive toxic metabolites. Thus, (I) the possible involvement of glial cells

in

neurodegenerative processes requires greater consideration that it has been

given in the past, and (II) changes in glial cell number and/or functions may

contribute to the only evident risk factor in neurodegenerative disorders

such as Parkinson's disease, namely aging.

THE ROLE OF MICROGLIA IN BRAIN INJURY

Wolfgang J. Striet

Department of Neuroscience, University of Florida, Gainesville, FL 32610

Recent research has resulted in a concept which views microglial cells as

immunocompetent cells indigenous to the central nervous system. As such,

microglia are known to be critical for phagocytosis, antigen presentation, and

cytotoxicity. At the same time, evidence has been accumulating which supports a

neurotrophic role of microglial cells, particularly in paradigms of acute

brain injury . This presentation will review work which has examined microglial

activation in various brain injury paradigms, including peripheral nerve

lesions, cerebral ischemia, and neurotoxicant-induced brain injury. The immuno-

and lectin histochemical observations will be discussed with reference to

proposed neurotoxic or neurotrophic functions of microglial cells.

FDA PROPOSED GUIDELINES FOR NEUROTOXICOLOGICAL TESTING OF FOOD CHEMICALS

T. J. Sobotka

Center for Food Safety and Applied Nutrition, Food and Drug Administration,

Washington, D.C.

That some chemicals adversely affect the nervous system is certainly not

news in regulatory toxicology. In 1982, the Food and Drug Administration (FDA)

issued testing guidelines for the safety evaluation of proposed direct food

and color additives. Assessment of nervous system toxicity was included as part

of the general toxicological profile. However, these guidelines provide only

broad and nonspecific recommendations for carrying out such an assessment.

Little more than the description of very apparent adult nervous system

toxicity associated with general neuropathology and overt neurological

dysfunction

can be derived from toxicity screening studies conducted according to these

guidelines. Little consistent or systematically documented information is

typically developed about other equally important types of neurotoxic effects

such

as behavioral dysfunction and developmental neurotoxicity. Concern about

these more subtle types of neurotoxicity has become a prominent public health

issue. This has resulted in demands for increased assurance that efforts are

being made to further minimize the risks of neurotoxicity from human exposure

to chemical substances. In an effort to address this concern, FDA is revising

its testing guidelines for food chemicals to include a more careful

evaluation of the structural and functional measures of neurotoxicity. These

are to be

routine components of safety assessment in adult and developing organisms.

Such a focus will generate the type of information needed for a more effective

assessment of the full spectrum of neurotoxic hazards. The revised

guidelines for neurotoxicity testing will be discussed in terms of FDA's

overall

approach to safety assessment.

Neuropathology in Risk Assessment of Xenobiotics

H. B.

Safety of Medicines Department, Zeneca Pharmaceuticals, Alderly Park,

Macclesfield, Cheshire SK10 4TG, United Kingdom

The fundamental requirements for registration of a new chemical entity are

that it should be efficacious and that exposure or administration to man

should prevent an acceptable risk. Evaluation of the safety of potential new

drugs

and chemicals (xenobiotics) is of critical importance in determining their

progression from industrial development to registration and subsequent

marketing. The responsibility for making this evaluation rests with the

manufacturer

and depends upon information derived from a diverse array of in vitro and in

vivo experiments in bacterial and mammalian species that are demanded by

Regulatory prescription. Assessment of the neurotoxicological phenomena

associated with exposure to certain xenobiotics differs substantially according

to

their type. An insecticide will be evaluated in stringent tests defined by

national agencies whereas a dopamine receptor agonist intended for treatment of

Parkinson's Disease may not. These significant difference in scientific

practices are incidental if the determination of product safety in animals is a

true predictor of its safety when given to man. Neuropathological assessment

represents a vital component of this neurotoxicological evaluation as it

provides qualitative and quantitative evidence of morphological perturbation

which

may be correlated with functional deficits. This evidence is pivotal in

weighing the risks against the benefits associated with treatment by or

exposure to

a xenobiotic. The focus of this presentation is to discuss the positive and

negative aspects of neuropathological assessments in animal studies and

address their benefits in prediction of potential human risk posed by

xenobiotics.

INVESTIGATIONS OF NEUROTOXICITY BEYOND SCREENING

L.A. Ivens

A. G. Bayer, Department of Toxicology, 42096 Wuppertal, Germany

The nervous system is an extremely complex " organ " with many subsystems

which all have to function to together. Other organs and tissues like the

heart,

muscle, kidneys, lung, and even the immune system are interconnected with the

nervous system. Xenobiotics may affect the whole nervous system or only

sub-systems like certain cell populations or even act indirectly through

effects

on other organs. Neurotoxicity screening batteries were developed to answer

the question: are effects of a novel compound seen which indicate impairments

of nervous system functions. Generally they are not able to answer specific

questions about the type of effect or the part of the nervous system which is

impaired. As soon as specific information about the area and type of change

in the nervous system is needed, this results in the need for specific methods

investigating specific parts and functions of the nervous system. The most

suitable method or methods have to be selected, since this type of specific

investigation is generally time consuming. Methods possible may vary as wide as

behavioral techniques, electrophysiology, histology and biochemistry. The

selection of methods depends on the area in which the neurotoxic is suspected

and/or most prominent. Further, information of the possible neurotoxicity of a

compound may also come from human case studies, from animal experiments or

from structural similarities with known neurotoxic compounds. Examples are

given of investigations of learning and memory by Maze and Active

Avoidance. Biochemistry, and electropysiology in vivo and in vitro are

discussed as

possible methods beyond screening tests.

AN APPROACH TO RISK ASSESSMENT

L. Simonsen, S.P. Lund and U. Hass

National Institute of Occupational Health, Denmark

A strategy for delineating risk factors from use of neurotoxic chemicals was

applied to the Danish working environment1 . This analysis disclosed the

need for internationally adopted criteria for neurotoxicity, and consequently a

working group was established by the Nordic Council of Ministers with the

task to suggest criteria for neurotoxicity2 . Effects on the nervous system,

e.g. reduction in memory and learning ability, decrease in attention, and

alteration of behavior due to toxic chemicals in the environment is now being

acknowledged as an important public health problem. This change in concern from

obvious effects of high dose exposure to the more subtle effects of

environmental exposure to neurotoxicants was incorporated in the criteria. The

approach

for evaluating neurotoxicity data suggested by the working group has

subsequently been used on 80 common industrial chemicals. The results indicate

that

numerous persons are exposed occupationally and in the environment in general

to several chemicals, for which almost no data on the effect on more subtle

neurophysiological functions are available. Development of a risk assessment

approach dealing with this problem is a major challenge of neurotoxicology in

the nineties.

1 Simonsen L, Lund SP: Amer J Ind Med 21:773-792, 1992

2 Simonsen L, sen H, Lund SP, Matikainen E, MidtgÜrd U, Wennberg A:

Scand J Work Environ Health 20:1-12, 1994

PROSPECTS FOR USING NEUROTROPHIC FACTORS IN THE TREATMENT AND PREVENTION OF

NEUROTOXICITY: AN OVERVIEW

S.C. Apfel

Departments of Neurology and Neuroscience, Albert Einstein College of

Medicine, Bronx, NY

Toxic neuropathy represents one of the most likely clinical settings for the

early therapeutic application of neurotrophic factors. These are proteins

which promote the survival, differentiation, and gene expression of specific

populations of peripheral and central neurons. Numerous pre-clinical studies,

in-vitro and in-vivo, have demonstrated the potential usefulness of growth

factors, and clinical trials for some of them are either underway or are being

planned. We plan to briefly review some of the basic biological features of a

variety of neurotrophic factors which may have particular importance in both

the pathophysiology and the treatment of toxic neuropathy. We will also

review the important pre-clinical studies, and assess their current status as

it

pertains to their potential clinical use. We will include discussions of

neurotrophin gene family members, ciliary neurotrophic factor, the insulin like

growth factors, and others.

THE DEVELOPMENT OF RECOMBIANT HUMAN NERVE GROWTH FACTOR

AS A TREATMENT FOR PERIPHERAL NEUROPATHIC DISEASE

B.C.

Genetech, Inc., South San Francisco, CA

Since its discovery over thirty years ago, the role played by nerve Growth

Factor (NGF) in the development and maintenance of sympathetic and selected

sensory neurons of the peripheral nervous system has been well established and

extensively studied. In addition, the existing data suggest that NGF may

offer neuroprotection to dorsal root ganglion neurons against drug- and

diabetes-induced neurotoxicity. Based on its neurotrophic and neuroprotective

activities, the systemic administration of NGF has been proposed as a treatment

for se

veral types of neurodegenerative diseases including diabetic peripheral

neuropathy where sensory and sympathetic neurons are affected early in the

course

of the disease. Until recently, painstaking purification and separation

techniques were required to obtained minimal quantities of NGF. The cloning of

the human gene and the subsequent large-scale production of recombinant human

NGF (rhNGF) has allowed for the initiation of comprehensive preclinical animal

studies and the initiation of human clinical trials. Growing evidence from

these and other studies suggest that the physiological actions of NGF may not

be strictly limited to the nervous system but may also include other organs

and systems. Results from recent preclinical studies also indicate no safety

findings which would preclude the chronic system administration of rhNGF in

clinical trials in humans. Results from on-going human trials with rhNGF should

greatly advance our understanding of the biology and therapeutic potential of

this molecule for treating peripheral neuropathic disease.

INSULIN-LIKE GROWTH FACTOR-I (IGF-I) PREVENTS THE PERIPHERAL NEUROPATHY

INDUCED BY VINCRISTINE, PACLITAXEL AND CISPLATIN

P. Contreras, C. Steffler, J. A. Gruner, S. C. Apfel, C. Brosnan, S. Dennis,

J. C.Arezzo, A. Yee, J. Kessler and J. L. Vaught

Cephalon Inc., WestChester, PA and Albert Einstein College Medicine, Bronx,

NY

IGF-I is a neurotrophic peptide that enhances the survival of motoneurons,

induces neuronal sprouting and enhances the rate of regeneration of damaged

nerves. To assess whether IGF-I can also prevent or ameliorate the neuropathy

induced by anti-tumor agents, IGF-I was administered daily to mice injected

with vincristine (1.7 or 2 mg/kg administered twice per week for 8-10 weeks),

cisplatin (10 mg/kg administered once per week for 16 weeks) or paclitaxel

(21.6 mg/kg administered daily for 6 days). Vincristine treatment produced a

sensorimotor neuropathy characterized by decreases in conduction velocity in

caudal nerves, decreases in gripping ability, alterations in gait, reduced

responses to noxious stimuli, an increase in the number of degenerating nerve

fibers and abnormalities in myelination in the sciatic nerve. IGF-I-treatment

prevented or ameliorated the effects of vincristine in a dose-dependent manner

with the dose of 1 mg/kg providing significant neuroprotection. A sensory

neuropathy was induced by 16 weeks of treatment with cisplatin or 6 days of

treatment with paclitaxel. Cisplatin treatment decreased the conduction

velocity

of caudal nerves, altered gait and reduced the ability to respond to noxious

stimuli. IGF-I (1mg/kg) administered daily prevented development of the

cisplatin neuropathy. A milder sensory neuropathy induced by paclitaxel was

characterized by reduced responses to noxious stimuli and concentrations of

calcitonin gene-related peptide in cervical DRG. Doses of IGF-I greater or

equal to

0.3 mg/kg prevented or ameliorated development of the paclitaxel neuropathy.

These data strongly suggest that IGF-I may be useful therapeutically in

preventing the peripheral neuropathy induced by a variety of anti-tumor agents.

PRECLINICAL PROFILE OF RECOMBINANT-METHIONYL HUMAN BRAIN-DERIVED NEUROTROPHIC

FACTOR (r-metHuBDNF): ALTERNATIVE ROUTES OF DELIVERY WITH THIS POTENTIAL

THERAPEUTIC FOR NEURODEGENERATIVE DISEASES

Carl P. LeBel, Ph.D.

Department of Toxicology, Amgen, Inc.

Amyotrophoic Lateral Sclerosis (ALS) is a progressive degenerative disorder

of motor neurons in the spinal cord, brain stem and motor cortex, and is the

most common motor nervous system disorder. Unfortunately, despite decades of

research on this disease, the etiology and pathogenesis of ALS remains

unclear. The well known survival-promoting actions of trophic factors in

various

neuronal cell culture systems essentially forms the foundation for the

rationale to use trophic factors in the treatment of neurodegenerative

disorders.

Brain-derived neurotrophic factor (BDNF) is a member of the nerve growth factor

(NGF) family, of which NGF is the prototype. Specificity and proof of concept

of the survival-promoting actions of BDNF in vitro have been demonstrated in

embryonic and neonatal motor neurons. Additionally, BDNF treatment has been

shown to prevent the decrease of cholinergic function in an adult rat facial

motor neuron lesion model. Recent evidence has also shown that systemically

administered BDNF slows the progressions of motor neuron degeneration in

transgenic Wobbler mice. Despite the limited utility of animal models to

predict

treatment for ALS, it is know that patients with this disease eventually

succumb to limb and speech paralysis, and finally to death due to respiratory

insufficiency and /or complications therefrom. Given the inevitably fatal

outcome

of ALS, combined with the database of the effects of BDNF on motor neurons,

a Phase I/II clinical study in patients with ALS was proposed. Preclinical

safety testing was undertaken in order to support the clinical research. The

preclinical development of BDNF, starting with in vitro data, and moving

towards the preclinical safety strategy used to support the clinical studies of

systemically administered BDNF to treat ALS will be presented. Additionally,

data generated in other preclinical studies with intrathecal delivery of BDNF

will be presented.

COMPUTERIZED BEHAVIORAL TESTING OF HUMANS

W. Kent Anger

Center for Research in Occupational and Environmental Toxicology

no abstract

NEUROTOXICITY TESTING: ARE WE ON THE RIGHT TRACK?

Abby Li

Monsanto Comapny - Environmental Health Laboratory

Speakers representing industry, public interest groups, government, and

academia will engage in a lively debate on strategies for neurotoxicity

testing.

This session will build upon and bridge two early sessions (Monday and

Thursday morning) and question some assumptions that may have been taken for

granted. Questions to be debated included: How big a problem is neurotoxicity?

Two

what extent to industrial chemicals contribute to this problems? Are

regulators proposing too much or too little testing? Are academic research

approaches

used in neurotoxicology inappropriate for regulatory testing or not used

enough? Come join us and voice your opinions. (Discussants included:

Mattsson, Werner Classen, Barbara McElgunn, G. Gilbert).

RELEVANCE OF NEUROTROPHIC FACTORS TO NEUROTOXICOLOGY

S. Barone Jr.

Neurotoxicology Division, HERL, U.S. Environmental Protection Agency, RTP, NC

This panel discussion is to inform and instigate discussion among

participants about the impact of advances in neurobiology of neurotrophic

factors and

the relevance to neurotoxicology. The topics to be discussed will be loosely

arranged around three general areas which are the role of neurotrophic factors

in, (1) development of the nervous system, (2) compensatory responses

toinjury, and (3) as a therapeutic strategy following neural injury or in

treatment

of neurodegenerative disorders. After a brief review of the biological role

of neurotrophic factors, a discussion will follow regarding what types of

neurotrophic responses may be indicative of injury and the possible utility of

these alterations as biomarkers of neural injury. This session is meant to be

informal and anyone interested in presenting pertinent data should contact the

moderator (B.Y.O.D.).

EFFECTS OF 1,3-DINITROBENZENE ON MITOCHONDRIAL FUNCTION IN CULTURED

ASTROCYTES

I.A. Romero, R.J. Rist and N.J. Abbott

Physiology Group, King's College, London, UK

1,3-Dinitrobenzene (DNB) is an industrial chemical causing

methaemoglobinaemia, testicular damage and neurotoxicity with primarily

glio-vascular lesions

(Romero et al., 1991, Neuropath. Appl. Neurobiol., 17, 498). Increased

glucose consumption and lactate production associated with intracellular

depletion

of glutathione, have been observed in primary cultures of astrocytes after

exposure to DNB over 24 h (Romero et al., 1995, Free Rad. Biol. Med., in

press). These changes have been related to oxidative stress damage due to

futile

redox cycling of the parent compound. DNB and/or its oxygen radical metabolites

may have a direct action on the normal mitochondrial respiratory activities

leading to changes in cell metabolism. The present study examined

mitochondrial function in cultured rat astrocytes following exposure to DNB.

During the

first 4 h of incubation with DNB, there was a dose-dependent increase in the

reduction of the tetrazolium dye MTT in cultured astrocytes, suggesting

either an enhanced mitochondrial enzyme activity or an increased mitochondrial

membrane permeability to the dye. After incubation with DNB for 24 h, MTT

reduction dose-response curves reflected cytotoxic effects at 2 mM DNB. There

was a

dose-dependent decrease in the uptake of Rhodamine 123, an index of

mitochondrial membrane potential, by astrocytes following exposure to DNB for 2

h,

with a threshold DNB concentration of 0.1 mM. Changes in cell autofluorescence

(exc. wavelength 340 nm, emission wavelength 400- 510 nm) were monitored at

37°C, as an index of mitochondrial NAD(P)H/NAD(P) ratio. Blockade of electron

transport by potassium cyanide (0.5 mM) induced an increase in cell

autofluorescence, while the uncoupler FCCP (20 µM) decreased the signal, as a

result

of increased oxygen consumption. Concentrations of 1 mM DNB induced a

reduction in the autofluorescence signal, indicating a decrease in mitochondrial

NAD(P)H/NAD(P) ratio. This effect could be related to cell metabolism since the

decrease in the autofluorescence signal after DNB exposure could not be

observed when astrocytes were incubated at 4°C. This results suggest that one

of the

earliest effects in DNB intoxication in astrocytes is mitochondrial

impairment, and supports the hypothesis that metabolism of the parent compound

by

futile redox cycling leading to the production of free radicals is involved in

DNB-induced neurotoxicity._

SENSITIVITY TO 2,5-HEXANEDIONE OF NEUROFILAMENTS IN NEUROBLASTOMA CELL LINE

SK-N-SH INCREASES DURING DIFFERENTIATION

E. Heijink1, P. A. Bolhuis2, G. B. van der Voet3, F. A. de Wolff 1

1Coronel Laboratory and 2Department of Experimental Neurology,

University of Amsterdam, Academic Medical Centre, 1105 AZ Amsterdam

3Toxicology Laboratory, University Hospital Leiden, 2300 BC Leiden, The

Netherlands

Chronic occupational exposure to n-hexane may cause peripheral neuropathy

via the metabolite 2,5-hexanedione (2,5-HD). The most characterizing features

are axonal swelllings containing aggregates of neurofilamental (NF) proteins

in the preterminal axon and nerve degeneration distally. Mechanistic studies

have demonstrated that 2,5-HD can react directly with the NF-proteins

resulting in covalent-crosslinking. However, this reaction is observed within

other

proteins as well and can not exclusively explain the high vulnerability of

NF-proteins for 2,5-HD. Using the human neuroblastoma cell line SK-N-SH in

culture as a model, we studied the direct effects of 2,5-HD on the distribution

of

the NF-proteins with immunocytochemical methods. Retinoic acid induced

differentiation into neuronal cells expressed by the outgrowth of processes and

the detection of NF-proteins in the majority of the cells. Cells were exposed

to 0- 10 mM 2,5-HD for 3 days. A concentration-dependent accumulation of

NF-proteins was detected as a spherical structure in the perikaryon. NF in the

differentiated neuronal cells were more susceptible to 2,5-HD than in

undifferentiated cells, as effects occurred at much lower 2,5-HD

concentrations. At

present the dynamics of NF-proteins during 2,5-HD exposure are examined in both

undifferentiated and differentiated cells using metabolic labeling studies.

These experiments may be helpful in determining which NF-features are

important in 2,5-HD induced neurotoxicity.

IMMUNOHISTOCHEMICAL LOCALIZATION OF NEURONAL AND GLIAL CALCIUM-BINDING

PROTEINS IN HIPPOCAMPUS OF CHRONICALLY LOW LEVEL LEAD EXPOSED RHESUS MONKEYS

Sigrid Noack1, Gisela burg1, Hellmuth Lilienthal2 and Gerhard Winneke

2

1Institute of Neuropathology, Freie Universität Berlin

2 Institute of Environmental Hygiene at the University of Düsseldorf,

Germany

The purpose of this study was to investigate the distibution of the neuronal

proteins parvalbumin, calbindin D28k, calretinin and the glial protein S100

in the hippocampus of lead exposed rhesus monkeys. It has been suggested that

lead may exert its toxic effects by perturbing the intracellular calcium

homeostasis. Lead is able to increase the intracellular Ca2+concentration and

can serve as a calcium substitute. Some calcium-binding proteins such as

calmodulin or renal and intestinal calbindin are capable of binding lead. We

tried

to find out a putative dose-dependent relation between long-term low level

lead exposure and the expression of the proteins investigated. Rhesus monkeys

were pre- and postnatally exposed to 600mg-300mg-0mg lead-acetate in diet for 9

years, as described by Lilienthal et al. 1986. Hippocampal paraffin sections

were stained for parvalbumin (PV), calbindin D28k (CB), calretinin (CR) and

S100 with immunohistochemical methods. The distribution of the neuronal

calcium-binding proteins was almost identical for the different exposure groups.

The most striking observation was a marked decrease of S100 immunoreactivity

of astrocytes in the high lead group. Considering a protective role against

high Ca2+concentration and Pb2+accumulation respectively the unchanged

expression of PV, CB, CR remains to be clarified. The apparent difference of

S100

expression supports the hypothesis, that glial cells are the main target of

lead

toxicity. The reduced expression may indicate a developmental retardation of

astroglia.

EFFECTS OF CHRONIC LOW LEVEL LEAD EXPOSURE ON THE EXPRESSION OF FIVE

DIFFERENT NMDA-SUBUNITS IN HIPPOCAMPUS OF YOUNG RATS: A mRNA IN SITU

HYBRIDIZATION

STUDY

S. Partl1, G. burg1, M. Hummel2, H. Herbst2, L. Altmann3 and

H.Wiegand3

1 Institute of Neuropathology, Freie UniversitÑt Berlin, Germany

2 Institute of Pathology, Freie UniversitÑt Berlin, Germany

3 Institute of Environmental Hygiene, DÅsseldorf, Germany

The purpose of this investigation was to study the effect of chronic low

level lead exposure on the mRNA expression of different n-Methyl-D-Aspartate

(NMDA) receptor subunits in the hippocampus of young rats. The effects of lead

were investigated in a total of seventy 17 to 20 days old male Wistar rats

exposed to lead during gestation and postnatally until weaning (postnatal day

20). The control animals were the offspring of and suckled by unexposed

mothers. Radioactive mRNA hybridization was performed with P33 labelled

oligonucleotides for the different subunits of the NMDA receptor channel

complex: NR1,

NR2A, NR2B, NR2C and NR2D. Frozen horizontal sections were used. The expression

of NR1 and NR2A in the CA1 region of the hippocampus was significantly

higher in lead exposed animals compared to controls. The differences in

expression

of NR2B were less pronounced. According to normal developmental

distribution, the expression of NR2C was restricted to cerebellum and thalamus.

The NR2D

expression was no longer to be detected. The increased level of NR1 and NR2A

expression may indicate an upregulation to compensate the functional

alteration by lead on the postsynaptic or presynaptic site. The anatomical and

developmental distribution of NR2D was in physiological range. There was no

indication for developmental delay in the postnatal time interval investigated.

The

overexpression of the NR1 subunit correlates with the increased MK 801

binding and the reduction of long term potentiation (LTP).

2.5-HEXANEDIONE ACCUMULATES GFAP-POSITIVE INTERMEDIATE FILAMENTS IN

NONNEURONAL CELLS IN IN-VITRO CELL CULTURES OF THE NERVOUS SYSTEM

W. GrÅning1, G. burg2, F. Boegner 3 and H. Altenkirch4

1Department of General Medicine and Nephrology, 2Institute of

Neuropathology, 3Department of Neurology, Klinikum lin, Freie

UniversitÑt

Berlin 4Department of Neurology, Spandau Hospital, Berlin, FRG

In organic solvent neurotoxicity 2.5-hexanedione (2.5-HD) plays an important

role as the most potent neurotoxic metabolite of n-hexane and 2-hexanone. It

has been the toxic agent in a wealth of experimental studies. The well

identified histopathological correlate of the axonotonic effect consists in a

massive accumulation of 10nm neurofilaments proximal to Ranvier's nodes. We

have

already shown, that nerve cell cultures of chick embryos exposed to 2.5-HD

develop specific neurotoxic alterations. Different nervous system compartments

displayed different vulnerability reflecting clinical conditions. Apart from

topical divergencies neurotoxicity depended on concentration of 2.5-HD in a

linear manner. However, neurons failed to develop classic neurofilament

accumulation. In contrast, we found gliatoxicity exceeding neurotoxicity

significantly. Due to high proliferation rates gliatoxicity occurred with

threshold

dose resulting in abrupt pancytotoxicity. Now we demonstrated by

immunohistochemistry for glial fibrillary acidic protein (GFAP) that

subpancytotoxic

concentrations produced juxtanuclear accumulations of intermediate filaments in

nonneuronal cells. The alteration observed indicates breakdown of

cytoarchitecture. These results corroborate the general character of

intermediate filament

pathology and cytoskeleton dysfunction under 2.5-HD exposure.

FACTORS DETERMINING THE SENSITIVITY OF THE BRAINSTEM TO GLIOVASCULAR DAMAGE

J. L. Holton, C. C. Nolan, A. Sale & D. E. Ray

Medical Research Council Toxicology Unit, Leicester, England

The gliovascular lesions produced in the brainstem by 1,3-dinitrobenzene

(DNB) can be unilaterally decreased in severity by reducing auditory input to

one ear (Neurotoxicol. 13:379-388; 1992). This suggests that ongoing sensory

input may modulate the brainstem damage caused by this agent. A total dose of

8-12g/kg of the antibiotic metronidazole given to 10 rats over 10-15 days

produced lesions with a similar morphology and topography to those produced by

DNB. They also developed asymmetrically in animals with a unilateral left

tympanic membrane perforation. Mean lesion severity scores (out of 3) were 0.5

:

1.1 in the left : right cochlear nuclei (p=0.016, Wilcoxon test), and 2.1 :

1.2 in the left : right inferior colliculi (p=0.008), suggesting that the

sensory modulation effect is not unique to DNB. In rats given DNB (3x 9 mg/kg

at

0, 4 & 24 hours), we examined the effect of increasing motor activity by

inducing tremor by co-intoxication with the pyrethroid bifenthrin (20 + 10

mg/kg

at 6 & 24 hours).This treatment produced a fine generalised tremor which began

12 hours after the first dose of DNB and lasted until 42 hours. The

bifenthrin significantly increased the severity of DNB damage in motor areas

such as

the cerebellar roof nuclei and red nuclei. As expected, bifenthrin alone

caused no morphological damage in the central nervous system at this (maximal

tolerated) dose. The red nuclei and cerebellar roof nuclei are major motor

areas

in the rat, and show increased glucose utilisation during pyrethroid-induced

tremor. We therefore conclude that changes in motor, as well as sensory

activity, can modulate the severity of these lesions.

MYELIN BASIC PROTEIN IN CEREBROSPINAL FLUID AS A MONITOR OF ACTIVE

DEMYELINATING LESIONS IN THE RAT CNS

X. Liu, P. Glynn & D. E. Ray

Medical Research Council Toxicology Unit, Leicester, England

A number of chemical agents produce direct myelin damage leading to

demyelination, and others produce secondary demyelination consequent on

axonopathy.

We hypothesised that myelin breakdown would lead to liberation of myelin basic

protein (MBP) into the extracellular space, and thence into the

cerebrospinal fluid (CSF). As CSF turnover is relatively rapid, then the

concentration of

MBP might provide a measure of the current status of a lesion (i.e. active

or inactive). In order to test this, rats were implanted with a chronic

catheter in the cisterna magna and concentrations measured in 100 µ1 samples of

CSF

by radioimmune assay using antibodies developed against purified rat MBP.

Following a test injection of MBP in 3 rats, CSF concentrations fell with an

initial half life of about two hours. The detection threshold was approximately

2 ng/ml MBP. Standard demyelinating lesions were created by microinjection

of 25 µg of lysolecithin in 2.5 µ1 saline bilaterally into the cerebellar

white matter. Lesions had an approximate diameter of 1.5 mm at 72 hours, and

were

characterised by extensive demyelination. They produced no obvious motor

signs, but cerebellar auditory evoked response amplitude decreased reversibly,

reaching a minimum at 3 days. The demyelination had largely resolved when

examined at 15 days. Prior to injection no MBP could be detected in CSF

samples,

but within 6 hours of lesioning MBP concentration reached 84±19 ng/ml,

remained elevated at 24 hours, and fell to 33±13 ng/ml at 2 days (n=5,

mean±S.E.).

MBP could be detected at 3 and 4 days, but not at 6 days or later. Rats given

control injections of saline developed only minor needle track artefacts and

no elevation in MBP (n=5). We conclude that measurement of MBP in CSF can be

used to monitor the progress of chemical demyelination, provided

sufficiently large CSF samples are available.

NEURONAL DYSFUNCTION ASSOCIATED WITH GLIAL LESIONS IN THE RAT BRAINSTEM

D. E. Ray, J. L. Holton, T. Lister, M. Mulheran and C. C. Nolan

Medical Research Council Toxicology Unit, Leicester, England

The astrocytic and oligodendrocytic death seen in brainstem nuclei following

systemic administration of 1,3-dinitrobenzene (DNB) is associated with vacuol

ation and perivascular údema. The údema resolves over 8 days, giving place

to astrocytic and microglial proliferation. These lesions can produce severe

ataxia and a degree of secondary neuronal death, but are primarily glial. We

investigated the relationship between lesion severity and neuronal function.

Eleven rats were implanted with platinum recording electrodes in the inferior

colliculi. Auditory evoked responses were measured in response to 40 kHz tone

bursts, and local blood flow was measured by hydrogen polarography using the

same electrodes, both before and over 8 days following administration of 30

mg/kg DNB. Lesion severity was assessed in rats killed by arterial perfusion

with fixative at 8 days. Lesions in the auditory pathway (cochlear nuclei,

superior olives and inferior colliculi) were scored on a qualitative 0-3 scale

based on the proportion of the nucleus involved. Overall auditory pathway

lesion score ranged from 0/9 to 6/9. Inferior collicular blood flow showed a

large increase of from 0.95±0.03 ml/g/min (mean ±S.E.) to a peak of 3.10±0.42

at

12 hours. This preceded development of lesions, which develop from 12 hours

onwards. Flow returned to predose values after 72 hours. The magnitude of the

flow increase correlated with the severity of the subsequent lesion as

assessed at 8 days, linear regression giving a correlation coefficient of 0.676

(p=0.022). The amplitude of the collicular auditory evoked response to 100 dB

stimuli was significantly depressed over 24 to 120 hours, but returned to

normal at 8 days. At 24 hours it was 49±7% of the predose value. The peak

auditory deficit was well correlated with the overall auditory pathway lesion,

linear regression giving a correlation coefficient of 0.756 (p=0.007), although

less well correlated with the local lesion in the inferior colliculus (r=0.627,

p=0.039). We conclude that morphological measures of gliotoxicity correlate

well with neuronal dysfunction, as measured in the auditory pathway.

LOW LEVEL EXPOSURE TO ORGANIC SOLVENTS IN RATS MAY INDUCE A LONG-LASTING

INCREASE IN THE AUDITORY EXCITABILITY

S.P. Lund, L. Simonsen and U. Hass

National Institute of Occupational Health, Denmark

Several studies demonstrate reduced auditory sensitivity and/or permanent

threshold shift in rats exposed to various organic solvents, e.g. toluene,

trichloroethylene, xylene, styrene. The hearing impairment is most pronounced

in

the mid-frequency range and is probably related to outer hair cell loss in

the cochlea. For toluene and trichloroethylene, the reduction in auditory

sensitivity is not detectable until the dose of exposure has exceeded a certain

threshold. However, less attention has been focused on changes in the auditory

function at low exposure doses, i.e. before the outer hair cells are lost and

the auditory thresholds are increased. To test the effect of organic solvent

exposure on the auditory function, rats were exposed to trichloroethylene

(100 ppm, 1000 ppm), n-heptane (800 ppm, 4000 ppm), p-cymene (50 ppm, 250 ppm),

and 4-tert.butyltoluene (40 ppm, 20 ppm) 6 hours/day for 28 days. Two months

after the end of exposure the rats were anesthetized and the auditory brain

stem responses to 25-95 dBlin SPL 16 kHz tonepips were measured in steps of

10 dB. The group exposed to 4000 ppm n-heptane showed a reduction in the

auditory sensitivity together with an increase in the auditory threshold, while

the auditory sensitivity in the groups exposed to 1007 ppm trichloroethylene

and 800 ppm n-heptane showed increase without shifts in the auditory

thresholds. These results demonstrate the possibility of long-lasting increase

in the

auditory excitability and suggest a bellshaped dose-effect relationship of

organic solvent exposure on the auditory function in rats.

ACUTE EFFECTS OF INHALED TRICHLOROETHYLENE (TCE) ON RAT VISUAL FUNCTION

DEPEND ON EXPOSURE CONCENTRATION AND NOT DURATION

W.K. Boyes and V.T.

Neurotoxicology Division, U.S.E.P.A., and ManTech Environmental Technology,

Research Triangle Park, North Carolina

Risk assessments traditionally have focused on averaged lifetime exposures,

and have not considered hazards of brief exposures. These experiments explore

the relationships between exposure concentration © and time (T) to provide

guidance for assessing the risk of short-term solvent exposures. The null

hypothesis was that the product of CxT would produce a constant health effect

(Haber's Law). Awake, adult, male Long- rats were exposed to TCE vapors

in a head-only exposure chamber while recording pattern onset/offset visual

evoked potentials (VEPs). Experiment 1 involved exposure to 0, 500, 1,000,

2,000 or 4,000 ppm TCE (n=5/concentration) and testing 0.5, 1, 2, and 4 hr

after

exposure began, for CxT products ranging from 0-16,000 ppm-hr. Results showed

a selective reduction in amplitude of the VEP frequency double component

(F2) related to C, but not to T or CxT product. Experiment 2 focused on CxT

products of 0 ppm-hr (0 ppm-4 hr) or 4,000 ppm-hr created through 4 exposure

scenarios: 1,000 ppm-4 hr; 2,000 ppm-2 hr; 3,000 ppm- 1.3 hr; or 4,000 ppm-1hr

(n=9-10/concentration). Again, the results showed a significant decrease in F2

amplitude as a function of C, but not T or CxT product. These limited results

imply that for short term exposure health risks, exposure concentration is

more important than exposure duration. The outcome may differ, however, for

other endpoints, compounds, or exposure scenarios.

QUANTITATIVE EEG AND CSF CYTOLOGY FINDINGS IN INDIVIDUALS WITH CHRONIC

OCCUPATIONAL EXPOSURE TO MIXTURES OF ORGANIC SOLVENTS

Kiti M I Muller Mari Antti-Poika and Tero Kovala

Finnish Institute of Occupational Health, Helsinki, Finland

Quantitative electroencephalography (QEEG) recordings and cerebrospinal

fluid (CSF) cytological analysis were performed in 70 subjects (63 men and 7

women) referred to our Institute for examinations because of significant

occupational exposure to mixtures of organic solvents and symptoms suggestive

of

organic brain disease. The mean age of the subjects was 49.4 years (median 51,

range 31-61yrs) and the mean/median exposure time 26 yrs (range 8-45yrs). The

EEG recordings were performed with Cadwell Spectrum 32 equipment. The

quantitative analyses were made within the frequency band of 1.5-20 Hz with the

Neurometrics program and reference values of our own laboratory. CSF

cytological

analysis was performed from Millipore filtration specimens and cytocentrifuged

cell smears as earlier described.1 An increase in the absolute or relative

power values of delta and theta EEG activity or a slowing in the mean

frequency of EEG was observed in 39 subjects (56%). CSF cytological

abnormalities

were found in 55 subjects (79%). The most frequently detected abnormality in

CSF

(in 36%) was an abnormal differential cell count with a predominance (>60%

of CSF cells) of mononuclear phagocytes, the presence of lipophages (37%) and

an increased proportion of enlarged stimulated lymphoid cells (>31% of

lymphoid cell population). Additionally the CD4+/CD8+ T cell ratio was elevated

in

the CSF of 14 subjects (22%). In only 8 subjects (11%) both the QEEG analysis

as well as their CSF finding were normal while 32 subjects (46%) were

abnormal for both their QEEG and their CSF cytological findings. Thus findings

indicative of diffuse brain dysfunction were detected in a large proportion of

subjects with chronic exposure to mixtures of solvents. 1 Muller et al. J

Neuroimmunol. 1990;30:219-227.

THE APPLICATION OF NEUROBEHAVIORAL TESTING METHODS TO THE OCCUPATIONAL HEALTH

CARE SETTING: THE EFFECTS OF TOLUENE IN WORKERS IN THE PRINTING INDUSTRY1

H.H. Emmen2, B.M. Kulig2, W.F.M. Rooijendijk3, J. Hofstee3, H. Muijser 2 and

J. Hooisma2

2 Department of Neurotoxicology & Reproduction Toxicology,

TNO Nutrition and Food Research, Rijswijk

3 The Occupational Health Service Midden Ijssel, Deventer, The Netherlands

Occupational exposure to organic solvents has been associated with a number

of neurobehavioral effects ranging from acute subjective feelings of

inebriation to severe and persistent changes in cognitive functioning and

personality

changes. Despite the growing evidence for solvent effects in occupationally

exposed workers, no generally accepted approach to worker monitoring has been

adopted. In the present study, neurobehavioral functioning was evaluated in

toluene-exposed printers tested in the context of routine physical

examinations conducted in the occupational health care clinic. The purpose of

the study

was to examine the effects of solvent exposure in workers exposed to a

single solvent and the exposure levels at which effects occur and to evaluate

the

feasibility of implementing neurotoxicity monitoring procedures in the

occupational health care setting. Onehundred and thirty one toluene-exposed

workers

and 69 non-exposed workers in the printing industry were tested using a

battery of automated neurobehavioral tests developed as part of the European

EURONEST project. The EURONEST test battery consisted of tests designed to

assess

changes in memory (digit spans forward and backwards), psychomotor slowing

(simple reaction time), attentional processes (color work vigilance test),

perceptual coding (digit symbol substitution), and visuomotor performance

(ballistic tracking). In addition, subjective symptoms were evaluated using a

standardized neurotoxic symptom questionnaire (NSC-60). Analysis of the symptom

questionnaire results indicated a higher prevalence of subjective complaints

related to memory difficulties, sleep disturbances, and sensorimotor changes

(paresthesias and reduced neuromuscular strength) in the group of

tolueneexposed workers. In addition, covariance analyses corrected for age and

educational

level also indicated a significant increase in simple reaction time in the

exposed group. These results demonstrate the feasibility of the application of

neurobehavioral testing techniques in the health care setting and suggest a

mild degree of psychomotor slowing workers with a mono-exposure to toluene.

Further evaluation of toluene-related effects using individual exposure data

is currently in progress in order to assess the significance of these results.

1 This work was supported, in part, by the Netherlands Ministry of Social

Affairs and Labour (SoZaWe) and forms part of the EC Concerted Action EURONEST

THE ROLE OF CONFOUNDING VARIABLES IN THE ASSESSMENT OF NEUROBEHAVIOURAL

EFFECTS OF CHRONIC SOLVENT-EXPOSURE

Ann M. on

National Institute of Occupational Health and Safety,Worksafe Australia,

Sydney, Australia

The presence and relative contribution of confounding variables is an issue

in the assessment of toxic effects on neurobehavioural function. Factors like

education level and alcohol use are well-known to exert effects on the

performance of neurobehavioural tests. In a prospective study of a cohort of

vehicle spray painters, measures of neurobehavioural function, demographic

characteristics, daily habits and general health status were taken from the

onset of

their exposure on four occasions for three full years. This study provided

the opportunity to assess the relative contribution of confounding variables

and the study factor, solvent exposure, on neurobehavioural function in a

study design in which the effects of confounding would be expected to be

reduced.

Multiple linear regression analysis was used with three confounding

variables, education level, alcohol use and occupational experience as well as

solvent exposure as predictor variables of neurobehavioural test performance.

Although the level of solvent exposure was considerably less than the composite

threshold limit value for the solvent mixtures encountered, the results

indicated that psychomotor performance deteriorated with increasing solvent

exposure, but only on the hand steadiness test. The confounding variables

showed even

greater influences on performance. Education level affected performance on

both psychomotor and cognitive tests in the expected direction. Occupational

experience also influenced test performance but only for psychomotor tests.

With increasing time in the trade, spray painters showed significantly superior

performance on reaction time, hand steadiness and visual pursuit tests.

Since training in spray painting focuses on psychomotor coordination, this

result

would be expected. Alcohol use was associated with mixed effects on

neurobehavioural function. Painters who reported using greater amounts of

alcohol

showed significantly poorer performance on the visual test, critical flicker

fusion, but better performance on the short term memory and learning measures

of

the paired associates test. These findings reinforce the importance of

separating effects due to confounders from effects due to toxic exposures in

studies of neurobehavioural function. They emphasise the dilemma of test

selection, choosing tests that are sensitive enough to detect effects due to

toxic

exposure but which are not affected by confounding variables.

PERIPHERAL MARKERS OF NEUROCHEMICAL EFFECTS AMONG STYRENE-EXPOSED WORKERS

E. Bergamaschi, A. Mutti, S. Cavazzini, M.V. Vettori, F.S. Renzulli and I.

Franchini

Laboratory of Industrial Toxicology, University of Parma Medical School,

Italy

Several parameters involved in neurotransmission represent selectively

vulnerable targets for environmental and occupational neurotoxicants. In order

to

evaluate possible changes in biochemical events occurring in the central

nervous system, monoamine oxidase B (MAO) and dopamine-b-hydroxylase (DBH)

activities were measured in accessible biological media during a

cross-sectional

investigation in workers occupationally exposed to styrene. The study group

consisted of 53 workers (33 men and 20 women) exposed to styrene, employed for

9.3 years on average (range 1-22) in reinforced plastics plants. On the basis

of indices of internal dose (i.e., the sum of styrene metabolites in the

" next morning " urinary samples) two subgroups of workers were identified, whose

median metabolite levels were 88.6 and 340.2 mg/g creatinine, respectively.

Sixty industrial workers with no known exposure to chemicals and comparable as

to age, sex and confounding variables were recruited as controls. The

activities of MAO in platelet-rich plasma and of DBH in serum were measured

within

the same run for both groups using methods based on HPLC-ECD. A lower DBH

activity was found in exposed as compared to control workers (GM: 10.11 U/ml

serum vs. 7.25 U/ml serum; p < 0.01), whereas MAO activity was affected in the

heavily exposed subgroup (13.8 vs. 10.1 U/107 platelets; p= 0.05). After

adjustement for confounding variables, DBH correlated with styrene metabolites

(R2=

-0.182, p=0.005); a dose-response relationship between styrene metabolites

and DBH activity was also apparent (c2 =13.3, p< 0.01). This study confirms

that long-term exposure even to relatively low levels of styrene can affect DBH

activity. Since DBH is expression of catecholamine firing, its decreased

activity could represent an indirect index of an altered turnover rate of the

physiological substrate (i.e. dopamine) at neuronal level. However, a direct

consequence of styrene metabolites on enzyme activity cannot be ruled out.

Platelet MAO B activity does not parallel that of DBH and seems to be less

sensitive to styrene exposure.

VARIATIONS OF URINARY CATECHOLAMINES AND EXPOSURE TO STYRENE

M. P. Sassine, G. Truchon, R. Savard, S. Bélanger & D. Mergler

Université du Québec a CINBIOSE; Institut de recherche en santé et en

sécurité du travail du Québec, IRSST

Styrene, a well-known neurotoxin, is frequently used in industry. Animal

studies suggest that styrene targets the catecholaminergic system. The aim of

this study was to examine pre-shift (Monday) and end-shift (Friday) variations

in urinary catecholamines in relation to exposure among actively employed

workers. The study population included 47 workers, chosen randomly, from three

Québec fiber glass reinforced plastics factories; 9 were excluded for the

following reasons: history of disease that could influence catecholminergic

functioning; consumption of medication or drugs; organic solvent exposure

outside

of working hours. The time weighted average (TWA) for environmental styrene,

measured for each worker in their respiratory area, varied between 666.0

mg/m3 and 8.0 mg/m3 (geometric mean: 100.1 mg/m3) and exposure was constant

throughout the week. The TWA of workers not wearing a mask was strongly related

to

end shift urinary metabolites (mandelic and phenylglyoxylic acids); the

summation of these metabolites was used as bioindicators for the level of

styrene

exposure. The level of catecholamines were adjusted for confounding factors

such as age, scolarity, consumption of coffee, cigarette and alcohol, and

weight. End-Shift dopamine levels decreased with increasing level o urinary

metabolites (r2=0.17; p=0.05), factoring in seniority. Monday to Friday

increase

of urine norepinephrine levels was significantly correlated to the level of

exposure (r2=0.20; p=0.01). On the other hand, changes of epinephrine were not

associated to the level of exposure. Further studies should examine the

relation between performance on neurobehavioral test batteries an

catecholamines

excretion in order to better understand the mechanisms that underlay

neurotoxic manifestations in humans.(This study was financed by grants from the

IRSST

#PE-92-06 and FRSQ-CQRS #92-0756).

SYMPTOM BASE TATES AFTER CHEMICAL EXPOSURE FOR WHITE, HISPANIC AND AFRICAN

AMERICANS

R.M. Bowler1, Guy Huel2 , D. Mergler3 , J.E. Cone4, S . Rauch1

1 San Francisco State University, CA

2 I.N.S.E.R.M., Paris, France

3University of Quebec, Montreal

4 University of California San Francisco, CA

The results of a symptom checklist of three matched-pair studies of exposed

groups of: 1) a primarily white community (N=220) environmentally exposed to

metam sodium, a toxic pesticide, 2) a Hispanic group who worked in a

microelectronics plant and had extensive past (M 6.7yrs) exposure to multiple

organic

hydrocarbon solvents (N=180); and 3) an African/American environmentally

exposed group (N=168) who had sulfuric acid exposure are presented. Each

exposed

group was compared to a matched (race, age + 3yrs, gender and education +

2yrs and the nearest number of children) unexposed control group, resulting in

90 matched pairs for the metam sodium group, 61 matched pairs for the organic

solvent group and 77 pairs for the sulfuric acid group. MANOVA results for

all three exposed and unexposed groups indicate no differences in symptoms

for: weight loss, heart palpitations, anxiety, depression, chest tightness,

increase or decrease in sleep, dark vision. Differences were obtained at

p=<.001

for: Neurological symptoms: tingling, muscle twitching, tingling, numbness,

tremors, loss in muscle strength in hands and feet, fainting, perspiring,

blurred vision and change in personality; at p=<.01 for: incoordination and

diarrhea; at p=<.05 for irritability, lower alcohol tolerance and loss of

smell.

MANOVA results indicate no significant differences between all three exposed

and control groups for age, race, education and gender. Significant

differences by education alone were obtained for weightloss, and for age on

depression

and irritability. Prevalence rates and odds ratios of symptoms will be

reported by race, exposure group and individual symptoms. These results suggest

a

robust symptom complex following chemical exposure and appear to generalize as

a specific symptom pattern regardless of specific chemical.

CLINICAL DATA ON THREE CASES OF OCCUPATIONALLY INDUCED PCB- INTOXICATION

H. Altenkirch1, G. burg2, D. Haller1, G. Walter1 , O. Hopmann

1 Department of Neurology, Spandau Hospital

2 Department of Neuropathology, lin Hospital Free University

Berlin, Germany

Since the ban of PCBs at the end of the 1970s, extensive measures have been

undertaken in Germany to dispose of PCB contaminated transformers. We report

on three patients with considerable skin exposure to PCBs, in particular to

Clophen A-30, while repairing or dismounting transformers. The periods of

exposition range from 4 and 5 to 20 years. All patients presented with distal-

symmetrical sensorimotor polyneuropathy as well as encephalopathy. One case of

hepatopathy was observed, chloracne occurred in all three cases. In two cases

the neurophysiological examination indicated an axonal, in one case a mixed

axonaldemyelinating neuropathy. A nerve- muscle biopsy revealed axonopathy as

well as chronic neurogenic muscular impairment. In one of the cases, the

neuropathy and encephalopathy progressed for a period of over 8 years after

termination of exposure. In the two other cases the neurological deficits

persisted over an observed period of 2-3 years. The reported clinical results

may

suggest that the long half- life of Clophen and its accumulation in fatty

tissue can lead to persistence of PNS and CNS impairment long after the period

of

exposure.

NEUROLOGICAL INVESTIGATIONS IN 23 CASES OF PYRETHROID INTOXICATIONS REPORTED

TO THE GERMAN FEDERAL INSTITUTE FOR HEALTH PROTECTION

H. Altenkirch, D. Hopmann, B. Brockmeier, G. Walter

Department of Neurology, Spandau Hospital, Free University Berlin

In 1993, 64 cases of chronic pyrethroid intoxications were reported to the

Federal Health Office in Germany. Shortly afterwards the media spoke of

thousands of cases of pyrethroid intoxications in homes. Twenty-three of the

persons reported were examined in a neurological department on an in-patient

basis

using clinical neurological, neurophysiological (NCV, EMG, VEP, AEP, SEP,

EEG), neuroradiological (CCT, SPECT, MRI) and laboratory investigations,

including the examination of pyrethroid values in blood and urine. The

pyrethroid

exposition involved carpets, moth killers, pesticide sprays and wood

preservatives. Nine of the cases presented with severe somatic disorders with

completely different clinical diagnoses, such as pituitary tumor, radiogenic

lumbosacral plexus paralysis, Guillain-Barre syndrome, spinal muscular atrophy,

with

no plausible relationship to exposition. Eight cases presented with multiple

chemical sensitivity syndrome (MCS) and normal somatic findings. In six of the

cases, the complaints could be attributed to pyrethroid exposition. There

was, however, not a single case in which evidence for irreversible PNS or CNS

lesions could be found.

PROMOTION OF ORGANOPHOSPHATE INDUCED DELAYED POLYNEUROPATHY: THE TARGET IS

NOT A PHENYL VALERATE ESTERASE

D. Milatovic, K.M. Osman, A. Moretto and M. Lotti

Istituto di Medicina del Lavoro, Universita degli Studi di Padova, Padua,

Italy

Certain esterase inhibitors exacerbate organophosphate induced delayed

polyneuropathy (OPIDP) and the effect is called promoion. Target for OPIDP but

not

for promotion is neuropathy target esterase (NTE). Although the target for

promotion shares some characteristics with NTE, it remains unknown (Moretto et

al., Toxicol Appl Pharmacol 1994, 129:133-137). Since NTE is a neural phenyl

valerate (PV) esterase (defined as PV activity resistant to 40 uM of the

non-neuropathic paraoxon and sensitive to 50 uM of the neuropathic mipafox;

, Arch Toxicol 1977, 37:113-115), the aim is to ascertain whether the

target for promotion is a PV esterase too. No correlation was found between the

ability of a given compound to promote OPIDP in hens and to inhibit

paraoxon+mipafox resistant PV esterases in brain and sciatic nerve. Therefore,

the

promotion site is not among paraoxon+mipafox resistant PV esterases. About 70%

of

total PV esterases from hen brain and sciatic nerve is not inhibited by

5xNTE I50 of non-promoter neuropathic inhibitors such as diiso-propylfluoro

phosphate (DFP, 5 uM) and mipafox (50 uM) (20 min, pH 8.0, 37 oC). Titration of

these PV esterases with the promoter phenylmethanesulfonyl fluoride (PMSF)

showed that a fraction (about 10%) was resistant to high concentrations of PMSF

(1 mM). Therefore, the promotion site does not belong to this fraction.

Inhibition by PMSF followed a first order kinetic. The calculated I50s for PMSF

were 50 uM in both brain and sciatic nerve, and the activity was 6,065+681 and

1,035+172 nmol of PV hydrolysed/min/g of tissue (mean+SD) in brain (n=19) and

peripheral nerve (n=11), respectively. However, other promoters such as L-(-)

and D-(+) methamidophos, and di-n-pentyl phenyl phosphinate, did not

significantly inhibit this enzyme (>10 mM for methamidophos isomers and 1 mM

for

di-n-pentyl phenyl phosphinate, same conditions as above). Moreover, no

inhibition of this enzyme in brain and sciatic nerve was found when the same

promoters were given to hens at effective doses (50 mg/kg p.o. for methamidophos

isomers, 5 mg/kg s.c. for di-n-pentyl phenyl phosphinate). We conclude that the

target for OPIDP promotion is not a PV esterase.

NEUROBEHAVIORAL FOLLOW- UP STUDY IN WORKERS EXPOSED TO MERCURY

W. G|nther1, B. Sietmann2, A. Seeber2

1 Health Office, Bitterfeld, Germany

2 Institute for Occupational Physiology at the University of Dortmund,

Germany

It is known that slight subjective symptoms could be observed after chronic

exposure to nearly 0.01 mg/m3 of in-organic mercury in the air, and

psychomotor dysfunctions after chronic exposure to concentration above 0.1

mg/m3. The

present follow-up study investigated effects due to chronic exposure to

mercury vapours between 0.06 mg/m3 and 0.1 mg/m3 in the room air. Exposed

workers,

included in the study after an average exposure of 12 years, were observed 3

times during a period of about 4 years. High-exposure (n = 19, 125 100 5g/l

mercury in urine), low- exposure (n = 39, 22 12 5g/l) and control (n=37,

non-exposed) groups were defined. Eighteen different scales concerning

neurotoxic

complaints and personality were considered for statistical analyses, as well

as 4 variables concerning memory performance and 19 for perceptive,

sensomotor, interference and balance performances. Partial correlations

(corrected by

age and verbal intelligence) among mercury in urine and dependent variables

showed no stable pattern within the correlation coefficients in all three

observations. Analyses of covariance (corrected by age and verbal intelligence)

detected group differences in one of the memory tasks, in which a decrease of

correct word reproductions corresponded to an increase of the exposure.

Finger tapping and finger dexterity are related to the exposure in the same

manner. For the other performance data, the neurotoxic complaints and

personality

traits no stable relations to the exposure could be established.

VISUAL CONTRAST SENSITIVITY DEFICITS IN BOHEMIAN CHILDREN

H.K. Hudnell1, I. Skalik2, D. Otto1 , D. House1 and R. Sram2

1 Health Effects Research Laboratory, U.S. EPA, Research Triangle Park, NC

27711

2 Regional Institute of Hygiene, Czech Republic

Visual contrast sensitivity (VCS) tests have been used successfully in

medical diagnosis and subclinical neurotoxicity detection. Recently, VCS was

measured in two studies of 2nd grade children in the Czech Republic. Study 1

compared children in standard schools and schools for the learning disabled

(LD).

Study 2 compared children in Teplice, an area in which soft-brown coal

combustion produced high levels of pollutants (e.g. Hg, As, SO2, NOx, and

aromatic

hydrocarbons), with children in an area of low air pollution, Prachatice. It

was hypothesized that perinatal exposure to the combustion products disrupted

neurological development (Sram, 1991). The VCS test (Stereo Optical Co.)

consisted of circular fields containing sinusoidal gratings at 8 contrast

levels

for each of 5 spatial frequencies (1.5-18 cycles/degree). Subjects indicated

orientation of the patterns by pointing left, up, or right. Visual acuity

and VCS were measured in each eye of 74 children in Study 1, and 323 children

in Study 2. Hair and urine samples were collected in Study 2 and analyzed for

Hg and As. Children attending schools for the LD scored significantly lower

than controls on VCS, whereas visual acuity was normal. The deficit was

greatest at mid- to high spatial frequency. In Study 2, significant VCS

deficits

were seen in exposed children at low to mid-spatial frequency, even though

visual acuity was slightly above control level. Regression analyses showed that

VCS had no relationship to As, but an overall significant negative correlation

with hair Hg. However, analyses by district showed that mean Hg

concentration was higher in control than exposed children, and that the

relationship of

VCS to Hg was significant only in the exposed population. The results of Study

1 indicated that behavioral VCS testing is practical in young children, and

suggested that vision may be compromised in LD children. In Study 2, VCS was

lower in children living in Teplice, but the causal agent(s) remains unknown.

Separate mechanisms are suggested by differences in Study 1 and 2 VCS

effects.

MECHANISM OF DITHIOCARBAMATES POTENTIATION OF 1-METHYL-4-PHENYL

-1,2,3,6-TETRAHYDROPYRIDINE (MPTP) NEUROTOXICITY

S. Bachurin1, N. Lermontova1, E. Shevtzova1, L. Petrova1, L. Solyakov1, T.

Serkova1, T. Singer2, R. Ramsay2

1Institute of Physiologically Active Substances, Chernogolovka, 142432,

Russia 2University of California, San Francisco, USA

A lot of dithiocarbamate (DTC) derivatives are widely used in industry,

medicine and agriculture. Recently it was shown that diethyldithiocarbamate

potentiates neurotoxic effect of MPTP, which induces parkinsonism-like

neurological disorder in human and in some laboratory animals. In the present

work we

have studied structure-activity relationships of DTC derivatives action on

neurotoxic effect of MPTP in vivo and on the the key steps in the MPTP

mechanism

of action; particularly, on monoamine oxidase (MAO) catalysed oxidation of

MPTP, yielding 1-methyl-4-phenylpyridinium (MPP) and the following interaction

of MPP with dopamine uptake system and with mitochondrial respiration chain.

Among DTC studied the highest potentiation effect in experiments in vivo vivo

was revealed for diisopropyl-, dicyclohexyl- and diethylderivatives, whereas

number of their close analogs show no influence on MPTP neurotoxicity. It

has been shown, that the potentiation of MPP-metabolite intracellular toxicity

by DTC was a result of mitochondria respiration alteration (inhibition and

disruption) and change in dopamine uptake system functioning due to DTC

derivatives membranetropic action.

SUMMARIZING THE RESULTS OF NEUROTOXICITY TESTING: PHASE ONE - DATABASE

DEVELOPMENT AND CHARACTERISTICS

K.M. Crofton1, W.F. Sette2 and D.O. Norris2

1 Neurotoxicology, U.S. EPA, Research Triangle Park, North Carolina

2 Office of Polution Prevention, Pesticides and Toxic Substances, U.S. EPA,

Washington, DC

In 1985 the Office of Toxic Substances of EPA promulgated a set of

Neurotoxicity Testing Guidelines, which were subsequently revised and published

in

1991 by the Office of Pesticide Programs. Under the auspices of test rules and

consent agreements for industrial chemicals, and data call-ins for pesticides,

companies have submitted over 100 studies that contain data collected

accord- ing to these guidelines. This presentation outlines an effort to

collate

and analyze these data. The first phase will be the construction of a database

that will allow tracking of datasets. The second phase will involve detailed

data entry (i.e., study results) and methodological analyses (e.g.,

meta-analysis). Progress to date includes an alpha version of a program to track

datasets, written in Microsoft Access®. The program tracks chemicals based on

the

CAS number, chemical name and EPA Chem Code number (for pesticides). Each

dataset is recorded in a datafile and contains information on the submitting

company, report title, authors, year and basic information on the testing

methodology (e.g., species, duration of exposure, test methods) and a simple

description of the results. Initial efforts have been limited to those datasets

containing Neurotoxicity Screening Battery data (e.g., functional observational

battery, motor activity and neuropathology). Future plans involve expansion to

other neurotoxicity related datasets (e.g., OPIDN studies) and publications

in the open literature.

EEG CHANGES CAUSED BY SUBCHRONIC LEAD TREATMENT IN RATS

L. Nagymajtenyi, H. Schulz, and I. Desi

Department of Public Health, Albert Szent-Gyoergyi Medical

University,Szeged, Hungary

Lead is a well-known neurotoxic compound causing characteristic symptoms in

central and peripheral nervous system in both acute and chronic intoxication.

During the last decades, chronic low-level environmental lead exposure has

become more important than the occupational one. Although there are some

functional neurological symptoms among the signs of poisoning, the effect of

lead

on the bioelectric processes in the central and peripheral nervous system has

not been properly investigated. Male Wistar rats were treated by gavage with

80.0, 160.0 and 320.0 mg/kg lead (in form of lead acetate) for 4, 8 and 12

weeks. EEG of the anaesthetized animals (1000.0 mg/kg urethane) was registered

with silver electrodes put directly on the primary somatosensory, visual and

auditory areas. EEG was on line analyzed by a numeric analyzer program

(yielding mean amplitude and frequency, EEG index and frequency band power

spectrum of the recorded waves) and by a Waterfall program. There were

dose-dependent changes of the investigated parameters which became more

expressed by the

end of the experiment. Mean amplitude of the EEG had a moderate change, while

changes in the frequency (mean value, frequency band power spectrum) were

stronger. On the basis of these data we suppose that the chronic, low level

exposure can influence electrophysiological activity within the human brain.

EFFECT OF SUBCHRONIC MERCURY EXPOSURE ON EEG OF RATS

I. Desi, L. Nagymajtenyi and H. Schulz

Department of Public Health, Albert Szent-Gyoergyi Medical

University,Szeged, Hungary

Due to its neurotoxic effect, acute and chronic mercury exposure causes

clinical symptoms in the central and peripheral nerve system. In the present

study, we investigated the changes in EEG of rats subchronically treated with

mercury. Male Wistar rats were given by gavage 0.4, 0.8 and 1.6 mg/kg Hg (in

form of HgCl2) for 4, 8 and 12 weeks. EEG of the anaesthetized animals (1000.0

mg/kg) was recorded by silver electrodes placed directly on the primary

somatosensory, visual and auditory areas. Parameters of the recorded EEG,

calculated by a numerical analyzer computer program, were: mean amplitude, mean

frequency, EEG index and power spectrum of the frequency wave bands. Changes of

these parameters, depending on the dose and length of the treatment, were

manifest for EEG index and power spectrum but amplitude changes were moderate.

No

clinical sign of mercury poisoning was seen. Based on the above observations

we suppose that the chronic low-level exposure can affect brain electrical

activity in animals and also in humans.

SENSITIVITY OF NEUROTOXICITY SCREENING METHODS: EFFECTS OF LEAD ON BEHAVIORAL

ENDPOINTS AND BRAIN LEVELS OF GLIAL FIBRILLARY ACIDIC PROTEIN1

B.M. Kulig, J.H.C.M. Lammers, E.M.G. Hoogendijk and K.J.v.d. Berg

Department of Neurotoxicology & Reproduction Toxicology, TNO Nutrition and

Food Research, Rijswijk, The Netherlands

Although the sensitivity of the developing nervous system to the effects of

lead exposure is well known, effects in the adult organism have not been

extensively studied. Very high exposure levels in humans have been associated

with encephalopathy and signs of peripheral neuropathy and in more recent

studies, reversible changes in peripheral nerve conduction velocity at present

day

occupational exposure levels have been described (Muijser et al., 1987). The

purpose of the present study was to evaluate the effects of lead in adult

animals and to obtain further information on the sensitivity of behavioral and

neurochemical assessment methods for detecting neurotoxicity. Four groups of r

ats were dosed with lead acetate at 0, 4, 8 or 12.5 mg/kg i.p., 5 days/week

for 4 weeks. Neurological and behavioral changes were evaluated using a

standardized functional observational battery and automated motor activity

assessment prior to exposure, at the end of the dosing period, and at 2 weeks

post-exposure. In addition, concentrations of glial fibrillary acidic protein

(GFAP)

were measured in discrete brain areas using ELISA-based techniques 4 weeks

following the termination of exposure. Results indicated that repeated lead

exposure resulted in dose-related decreases both in the pattern and amount of

motor activity, decreased rearing, signs of motor dysfunction, and changes in

arousal. Dose-related increases in GFAP of up to 100% of control values were

also found in frontal and occipital cortex, hippocampus and striatum. Taken

together, these results demonstrate the sensitivity of these end-points for

detecting relatively low levels of lead exposure in adult rats. 1This work was

supported by the Netherlands Ministry of Social Affairs and Labour (SoZaWe)

and the Netherlands Ministry of the Environment (VROM).

SOCIAL PLAY BEHAVIOR IN JUVENILE RATS AFTER IN UTERO OPIOID EXPOSURE TO

MORPHINE

J.M. Niesink Louk Y.M.Y. Vanderschuren and Jan M. van Ree2

1Faculty of Natural Sciences, Open University, Heerlen and

2Department of Pharmacology, Rudolf Magnus Institute for Neurosciences,

Faculty of Medicine, Utrecht University, Utrecht, The Netherlands

Changes in analgesia, play behavior, sexual behavior and responsiveness to

stress and stimualants have been reported in rodents treated in utero with

opiates. During development the endogenous opioid receptors are present in a

tonic balance in the mammalian nervous system. The development of this balance

is particularly sensitive to prenatal administration of opioid agonists and

antagonists. The motivational and rewarding aspects of play behavior are

controlled by endogenous opioid systems; low doses of opioid agonists stimulate

play behavior, whereas administration of opioid antagonists inhibit play

behavior. We have analyzed the effect of morphine during the prenatal

development of

endogenous opioid systems by examination of play behavior in juvenile rats.

The doses of morphine used neither affected gestation of pregnant mother rats

nor motor- or sensoric development of the juvenile rats. Levels of pinning

and social grooming behavior in juvenile rats were elevated after prenatal

opioid exposure to morphine. To study these changes in more detail, social play

was investigated using a sequential analysis in prenatally morphine- and

saline-exposed pairs.

GLUCOCORTICOIDS ENHANCE OXYGEN RADICAL ASSOCIATED NEUROTOXICITY

L.J. McIntosh and R.M. Sapolsky

Department of Biological Sciences, Stanford University, Stanford, CA

Modern populations are constantly exposed to a variety of compounds in the

workplace and the environment that promote formation of reactive oxygen

species (ROS) within susceptible tissues. Due to its high oxygen consumption,

the

brain may be particularly vulnerable to oxidative damage and degeneration.

Agents that impact cellular oxidative homeostasis would therefore be expected

to

alter the toxicity of ROS generating compounds. We are testing this

hypothesis using endogenous stress hormones, glucocorticoids, to perturb

neuronal

homeostasis, and adriamycin to generate oxygen radicals. Glucocorticoids (GCs)

are hormones secreted by the adrenals in response to stress, and are also

prescribed clinically to control inflammatory and autoimmune disorders in

millions of people annually. Therefore, high GC levels may not be uncommon in

individuals exposed to low levels of toxic compounds. Also, GCs appear to act

on

cellular pathways relevant to ROS, as seen by their potentiation of

neurodegeneration following insults such as stroke, hypoglycemia, or seizure.

Using rat

primary neuronal culture, we determined neuronal susceptibility to adriamycin

toxicity by cell counting (using MAP-2 staining) and biochemical correlates.

Dichlorofluorescein fluorescence confirmed ROS generation after adriamycin

administration. Physiological levels of GCs (up to uM concentrations) in the

culture media exacerbated adriamycin toxicity. Further, we assayed antioxidant

enzyme activities from brain regions of rats either GC supplemented, or

adrenalectomized to eliminate circulating GCs. These tests showed that GC level

affected enzyme activity, and the pattern of difference was unique for each

enzyme. Our results indicate that stress hormones may directly affect pathways

involved in oxygen radical toxicity, and studies of these pathways could

indicate points at which antioxidant intervention would be useful.

MICROGLIAL RESPONSIVENESS AS A SENSITIVE MARKER FOR HEAVY METAL NEUROTOXICITY

F. Monnet-Tschudi, M.G. Zurich, E. Pithon, B. Pardo and P. Honegger

Institute of Physiology, University of Lausanne, Lausanne, Switzerland

In the search for early markers of neurotoxicity, the responses of

microglial cells, macroglial cells and neurons were analyzed in vitro, using

aggregating brain cell cultures of fetal rat telencephalon as a model.

Aggregates were

treated during an early developmental period with concentrations of

trimethyltin (TMT) at or below the limit of cytotoxicity. Microglia were found

to be

the most sensitive cell type, since already at 10-9 M of TMT an increased

number and clustering of Griffonia simplicifolia-positive cells could be

observed. At 10-8 M of TMT, an increased staining for glial fibrillary acidic

protein, characteristic of a gliosis, was found; as well as a decrease in

synaptic

proteins (synapsin I and synaptophysin) content in synaptosomal fractions. A

decrease in neuron-specific enzymes was observed at the highest concentration

tested, 10-6 M of TMT. Microglia may be the first target of TMT and their

reaction may trigger the responses of other cell types. On the other hand, it

cannot be excluded that discrete neuronal changes could act as an activating

signal for microglial cells. A similar sensitivity of microglial cells was

found after treatment with other heavy metal compounds, e.g., lead acetate,

mercury chloride, and monomethylmercury chloride. The present findings show

that

microglial responsiveness can be detected prior to any sign of neuronal

degeneration, and therefore may serve as a sensitive indicator for heavy metal

neurotoxicity in the brain.

ATTENUATION OF DOPAMINERGIC ACTIVITY IN NUCLEUS ACCUMBENS OF RATS EXPOSED TO

LEAD

A.L. Jadhav and S.V. Kala

Department of Pharmaceutical Sciences, College of Pharmacy & Health

Sciences, Texas Southern University, Houston, Texas USA

Long- hooded (21-day-old, male) rats were exposed to either sodium

acetate (control) or 50 ppm lead acetate in deionized distilled water for 90

days

and tissue contents of dopamine (DA) were studied in nucleus accumbens (NA).

Pb measurements were carried out using atomic absorption spectrophotometry

equipped with graphite furnace while DA was measured with HPLC coupled with

electrochemical detection. The exposure protocol produced blood Pb levels of

18+2 mg/dl, brain Pb levels of 350+20 ng/g wet tissue; and resulted in

significant reduction in DA contents (50%) in NA. These studies were extended to

examine whether the reductions in DA contents were associated with alterations

in

basal and stimulus (100 mM K+) induced release of DA in this brain region

employing the microdialysis technique. After the 90-day exposure period, rats

were anesthetized with urethane and 2 mm microdialysis probes were implanted in

NA (AP 2.7 mm, L 1.5 mm, V -7.5 mm) using a stereotaxic apparatus and

perfused with artificial CSF (aCSF) at a rate of 2 ml/min. The perfusates were

analyzed for DA. Consistent with the reductions of DA contents observed in Pb

treated rats the microdialysis studies indicated significant reductions in

basal

and potassium induced release of DA in Pb treated rats. These results

indicate that subchronic exposure to Pb results in reduced dopaminergic

activity in

NA. Supported by ATSDR/MHPF Grant No. U50/ATU398948-03.

NEUROBEHAVIORAL PERFORMANCE OF 4TH-GRADE CZECH CHILDREN LIVING IN DISTRICTS

WITH VARYING LEVELS OF AIR POLLUTION

D. Otto1, I. Skalik2, H.K. Hudnell1, D. House1, R. Sram2

1Health Effects Research Laboratory, U.S. EPA, Research Triangle Park, NC

2Regional Institute of Hygiene, Prague, Czech Republic

Northern Bohemia is one of the most polluted areas in Central Europe.

Elevated ambient levels of SO2, NOx, PAHs and heavy metals occur as a result of

intensive mining and combustion of brown coal for power generation. Sram (1991)

hypothesized that in utero exposure to these chemicals causes functional

changes in the nervous system expressed as developmental disorders or

behavioral

dysfunctions. Finger tapping performance was better in 8th-grade children

from the reference district of Prachatice than the mining district of Teplice.

Weak associations of hair mercury and neuro-behavioral performance were

observed in 2nd-grade children from these districts. 7 tests from the

Neurobehavioral Evaluation System (Letz 1991)--finger tapping, hand-eye

coordination,

continuous performance, visual digit span, symbol-digit substitution, pattern

comparison and switching attention--were administered to 504 4th-grade children

(165 from Teplice, 161 from Prachatice; and 178 from Znojmo, a district where

brown coal is not used). Children from Prachatice and Znojmo performed

better than children from Teplice on coding (p=.001) and digit span (p=.018)

tests. In conclusion, evidence of poorer neurobehavioral performance has been

found in several cohorts of children living in a heavily polluted mining

district

of Northern Bohemia.