INA 6 - Talks

O-1 HOLISTIC APPROACH OF NEUROTOXICOLOGY Szilárd, J. Department of Neurology and Psychiatry, Albert Szent-Györgyi Medical University, Szeged, Hungary

At the end of the 20th century we can say that the enthusiasm and expectations about natural sciences that was present at the end of the 19th century in many respects have not been fulfilled. There are too many dangers created by man and/or science. Storage of toxic waste, variability, mutation of pathogens, pharmaca, chemicals, radiation, and so on. There is a really wide scale where these cosmic, ecological, technical and human factors changes of climate etc. combine in front of our eyes. All these have a reasonable effect on the morbidity and mortality scales. In many respects it is the consequence of the harmful effects created by man itself and modern science that the biologically possible extension of human life and health including the sound function of the nervous system which was also made possible in theory by modern science never became reality. The most common and most vulnerable target organ of the exogenous dangering effects is the nervous system.

O-2 CONTRIBUTION TO NEUROTOXICITY OF ARSENIC IN ENVIRONMENTAL SETTINGS Bencko, V. Institute of Hygiene and Epidemology, First Faculty of Medicine, Charles University of Prague, Czech Republic

With the framework of a study concerning health consequences of human exposure to emissions rising from horning coal of high arsenic content (900 -1500 g per metric ton in dry substance) neurotoxicity of arsenic was one of the subjects of a special interest. Hearing changes were analysed after audiometric and clinical examinations of a group of 5610-years-old children (30 boys, 26 girls) residing near a power plant burning local coal of high arsenic content. The results obtained were compared with those of a control group 51 children (26 boys, 25 girls) of the same age living outside the polluted area. The highly standardized audiometric and clinical examinations were completed with a questionnaire analysis concerning the personal medical histories of the children. The data obtained were elaborated statistically by means of the c2 test. Significant hearing losses severe detected at frequencies of 125, 250 and 800 Hz at the both air and bone conduction examinations. While an assumed influence of the anamnestic presence of middle ear inflammation was fully documented in the control group, such does not appear in the exposed group. The high statistical significance of hearing losses at high frequency range (8000 Hz) in exposed group, (mean value 3,8 mg/g As in hair samples, range 0,86-10,33 mg/g ) are suspected to be of neurotoxic origin and bound points to the very low probability of their being only an accidental finding.

O-3 NEUROTOXINS AND NEURODEGENERATIVE DISORDERS: PARKINSON'S DISEASE Vécsei, L., Dibó, Gy., Kiss, Cs. Department of Neurology, Albert Szent-Györgyi Medical University, Szeged, Hungary

There is a hypothesis that excitotoxic pathomechanisms underlie neuronal tissue degeneration in disorders such as Parkinson's disease (PD), Huntington's disease (HD), amyotrophic lateral sclerosis (ALS) and dementia of the Alzheimer's type (AD). It is proposed that target neurons are overexcited, the result being energy disturbance and pathobiochemical changes that culminate in nerve cell death (see review by Vécsei et al. 1992). Diminished energy supplies may result either in excessive release of glutamate or an inability of the neuron to restore ionic balance after stimulation. The methyl-phenyltetrahydropyridinum ion (MPP+), the active metabolite of MPTP, selectively enters dopaminergic neurons via the dopamine-uptake system and inhibits the formation of Complex I of the mitochondrial respiratory chain. There are tardive neurologic disorders associated with nitropropionic acid (NPA), a suicide inhibitor of succinate dehydrogenase, carbon monoxide (CO), which binds to haemoglobin and heme proteins (including cytochrome oxidase), and formate poisoning from methanol intoxication. Parts of the basal ganglia appear to be especially vulnerable in energy-deficient states. Cassavism (CN poisoning) is characterized by a subacute onset of leg weakness and spastic paraparesis presumably associated with degeneration of the corticomotoneuronal pathway (see review by Spencer et al. 1992). There is the possibility that Mn causes cell damage by inducing mitochondrial toxicity and a consequent bioenergetic defect. This could impair the voltage-dependent magnesium blockade of NMDA receptors and render cells vulnerable to the excitotoxic effects of glutamate and other excitatory amino acids (Brouillet et al.1993; Olanow et al.1996).

O-4 THE NEUROTOXICITY OF POLYCHLORINATED BIPHENYLS(JACOB HOOISMA MEMORIAL LECTURE) Tilson, Hugh A., Kodavanti, Prasada R.S. U.S. EPA, Research Triangle Park, USA

Like dioxin, some PCB congeners produce toxicity by binding to an aryl hydrocarbon (Ah) receptor. Other PCB congeners that have little or no activity at the Ah receptor have been shown to appear in the brain following in vivo exposure and decrease dopamine content. Subsequent research has found that non-dioxin-like PCBs also interfere with calcium homeostatic mechanisms and intracellular second messenger processes in vivo. The biological significance of the effects of PCB in the nervous system preparations is not known, although there are a number of calcium-dependent processes that are important for nervous system function and development. SAR studies based on measures of PCB-induced alterations in protein kinase-C (PKC) translocation and Ca2+ -buffering indicate that congeners with chlorine substitutions at the ortho-position are active in vitro, while non-ortho congeners are relatively inactive. Subsequent research has found that chloride substitution patterns that favour non-coplanarity are associated with activity in nervous system preparations. Recent studies in vivo have shown that repeated exposure to a PCB mixture Aroclor 1254 increases translocation of PKC and decreases Ca2+ buffering in the brain. These changes in vivo are associated with elevated levels of ortho substituted non-coplanar PCB congeners in the brain. Current research is focusing on the possibility that PCB-induced alterations in calcium homeostasis and intracellular second messengers may be related to the developmental neurotoxicity of PCBs.

O-5 CENTRAL AND PERIPHERAL NEUROTOXIC EFFECTS OF CADMIUM IN CHILDREN AND ADULTS: A REVIEW OF THE LITERATURE. Viaene, M.K. Catholic University of Leuven, Leuven, Belgium

Since man started with the industrial production of cadmium in 1829, it soon became an occupational hazard, and more recently also an environmental pollutant endangering public health. Since the beginning, reports on cadmium toxicity are almost solely focussed on pulmonary, liver, bone and kidney effects, with the kidney being regarded as the most important target organ. Although it has been demonstrated many times in experimental situations that cadmium has major neurotoxic potentials, there seems to be little enthusiasm to search for human evidence of this neurotoxicity. This is probably due to early animal data, which showed that only small amounts of cadmium could pass the Blood-Brain-Barrier in adult animals, even under chronic exposure conditions. Nevertheless, in humans many sites in the central and peripheral nervous system are known to have less tight or even no barriers, e.g. the neuromuscular junction, the dorsal root ganglia, the autonomic ganglia, the optic nerve, the olfactory bulb and the circumventricular organs, which are part of the limbic system. Moreover, the effectiveness of the BBB can decline with ageing and the effect of cadmium on the nervous system could very well change with older age as also with younger age and nutritional status. This might have raised the suspicion that subpopulations of workers or some parts of the nervous system could be more susceptible to the deleterious effects. Reviewing the literature, we found eleven publications regarding cadmium neurotoxicity in occupational or environmental exposed persons and six studies on neurobehavioural effects of heavy metals including cadmium in children. Congruent to the experimental data, there are some studies and reports indicating an increase of anosmia and the existence of peripheral polyneuropathy in adults. Maybe more surprisingly, there is also evidence that cadmium exposure might not only give central nervous system effects in children, but also in occupationally exposed workers (EEG-alterations, tremor and neurobehavioural changes). The literature will be reviewed to look if further research is warranted.

O-6 NEUROBEHAVIORAL ALTERATIONS FOLLOWING EXPERIMENTAL CADMIUM-EXPOSURE: DIRECT OR INDIRECT NEUROTOXICITY? Winneke, G. Medical Institute of Environmental Hygiene, Heinrich-Heine-University, Düsseldorf, Germany

Neurobehavioral toxicity of cadmium has been shown for a variety of endpoints following gestational, neonatal and adult exposure of rats (see review of Hastings, 1995). Yet, any clear-cut interpretation of such findings in terms of a direct action of Cd on nervous tissue is obscured by the fact that very little cadmium crosses the placenta or the blood-brain barrier, respectively. It is only during the neonatal stages of development that sufficient amounts of Cd may enter the CNS to produce morphological and/or functional damage. Therefore, part of the observed Cd-related behavioral toxicity, particularly following gestational exposure, may be indirect, namely due to Cd-induced placental dysfunction, whereas there is a potential for more direct nervous system toxicity subsequent to early neonatal exposure. Anyhow, loss of body weight, often associated with Cd-exposure, interferes with any straightforward interpretation of observed neurobehavioral alterations in terms of direct neurotoxicity.

O-7 EXPERIMENTAL NEUROTOXICOLOGY OF CADMIUM: LESSONS LEARNED FROM IN VITRO STUDIES Wiegand, H. Medical Institute of Environmental Hygiene, Heinrich-Heine-University, Düsseldorf, Germany

Although many data suggest a high neurotoxic potential of cadmium the underlying mechanisms of neurotoxicity are far from being understood. There seems to be a striking discrepancy between the high susceptibility of some in vitro preparations of the nervous system for cadmium on one side and the apparently low impact of cadmium if exposure of the adult nervous system in vivo occurred. Several properties of cadmium neurotoxicity were elaborated for different transmitter systems, ion channels, and receptor types. Cadmium disrupted many neuronal processes and had many adverse effects in different systems by a variety of mechanisms. Integrated approaches seem to be necessary to further elucidate the widespread mechanisms of cadmium neurotoxicity.

O-8 TOXICOKINETICS AND BIOCHEMISTRY OF CADMIUM WITH SPECIAL EMPHASIS ON THE ROLE OF METALLOTHIONEIN Jin, T. Lu, Y.*, Nordberg, M.** Deptartment of Occupational and Environmental Medicine, Umea University, Umel, Sweden, *Department of Occupational Health, Sanghai Medical University, Sanghai, P.R. of China, **Institute of Environmental Medicine, Karolinska Institutet, Stockholm, Sweden

Cadmium is a toxic metal with extremely long biological half-time of 15-20 years in humans. Adverse health effects develops upon exposure. Renal tubular failure occurs after exposure and thus the kidney is regarded as the critical organ independent of exposure route. Cadmium is classified as a human carcinogen to cause lung cancer after inhalation. Effects on fetus is likely due to accumulation of cadmium in the placental tissue. Neurotoxicity of cadmium needs to be further evaluated as cadmium previously was not believed to pass the blood-brain barrier. There is some evidence that cadmium can be taken up in the central nervous system of suckling animals. Such uptake is modulated by induction of metallothionein by ethanol. Why cadmium does not pass placenta and to a less degree blood brain barrier might have several explanations involving metallothionein and interaction between zinc, copper, selenium and calcium with various receptors. The toxicokinetics of cadmium involves metallothionein - a protein with molecular weight of 6500 Da and rich in cysteine and with 7 metals distributed in two clusters. The most well studied metallothioneins refer to as metallothionein I and II with a quite a number of subforms expressed in almost all tissues. Metallothionein III is present in brain and IV is expressed in keratinocytes. Most dominating metals are cadmium, mercury, zinc and copper. Human fetal liver contains metallothionein rich in copper. The brain metallothionein is rich in zinc. Brain metallothionein has somewhat different characteristics and was thus not identified until an other electrophoretic technique was used. A review of current knowledge of the subject including mechanistic discussion will be presented.

O-9 THE EFFECT OF CADMIUM ON THE HIPPOCAMPAL ACTIVITY IN RATS. COMPARISON WITH OTHER TOXIC HEAVY METALS Papp, A., Nagymajtényi, L., Dési, I. Dept. Public Health, Albert Szent-Györgyi Medical University, Szeged, Hungary

Cadmium is one of the well-known toxic heavy metals emitted into the environment as a consequence of industrial use and its presence in different types of waste. Due to that, occupational as well as food- and water-borne cadmium exposure has happened in numerous cases and still happens. Cadmium has been known to affect mainly the kidneys, the bones and the cardiovascular and respiratory system. In the last years, however, a growing body of evidence points to its neurotoxic effect and emphasizes the need for studying the underlying mechanisms. In our studies, the effect of acute cadmium treatment on the in vivo hippocampal population spike in rats was investigated and compared to effects of lead and mercury (both known to be neurotoxic). The hippocampal population spike was chosen as a phenomenon with well-described structural and functional background which more or less directly reflects the activity of neurons. The amplitude of the population spike was increased by an acute high dose of cadmium, but there was hardly any change in its latency and duration. Lead, on the contrary, caused a significant amplitude decrease while the effect of mercury was (at ca. isotoxic doses) indefinite. Having these results, we investigated the effects of cadmium and lead on the tetanic potentiation of the population spike. Following a tetanus, cadmium left the increased population spike amplitude unchanged or caused a further increase while lead reduced the increase and diminished or abolished the effect of a second tetanization. Our results show that the in vivo hippocampal population spike in rats can be used to study the neurotoxic effect of cadmium. Supported by the Hungarian OTKA grant No. T016735

O-10 INSECTICIDES ON GABA- AND GLYCINE-DEPENDENT CHLORIDE FLUX Sunol-Esquires, Cristina, Vale, Carmen, Rodríguez-Farré, Eduard* Department of Neurochemistry and *of Pharmacology and Toxicology, Institut d'Investigacions Biomédiques de Barcelona, CSIC, Barcelona, Spain

The inhibitory neurotransmitters g-aminobutyric acid (GABA) and glycine directly cause an increase in conductance to Cl- by binding to ligand-activated ion channel receptors at the postsynaptic membranes, so that opening of Cl- channels usually leads to a net hyperpolarization. The GABAA receptor has separate but allosterically interacting binding sites for GABA, benzodiazepines, barbiturates, anesthetic steroids and the convulsant picrotoxinin. The GABAC receptor also forms a Cl- channel, however its pharmacology differs from that of the GABAA receptor. Neurotoxic organochlorine pesticides belonging to the group of polychlorocycloalkanes (cyclodienes and g-hexachlorocyclohexane or lindane) induce in mammals an hyperexcitability syndrome that can progress until the production of tonic-clonic convulsions. They act as non-competitive GABA antagonists interacting with the picrotoxinin site both in membranes and in intact cultured neurons and inhibit the GABA-induced Cl- flux following activation of either GABAA or GABAC receptors. We also report the effects of polychlorocycloalkanes on glycine-induced Cl- flux in primary neuronal cultures. The d isomer of hexachlorocyclohexane is a depressant compound. It increases the GABA-induced Cl- flux and allosterically increases benzodiazepine binding at the GABAA receptor. We discuss the mechanism of action of these compounds in relation to the disruption of ligand-operated Cl- channel receptors and the relevance for their convulsant/depressant action. Supported by FIS (97/0656) and CIRIT (SGR 95-00445; 95-00551). C.V. is recipient of a predoctoral fellowship from CIRIT, Generalitat de Catalunya.

O-11 PYRAZOLINE INSECTICIDES ON CALCIUM UPTAKE IN RAT BRAIN SYNAPTOSOMES Nishimura, Keiichiro, Tada, Toshiji Research Institute for Advanced Science and Technology, Osaka Prefecture University, Sakai, Osaka 593, Japan

Pyrazolines are a class of broad-spectrum insecticides. Methyl 3-(4-chlorophenyl)-1-[N-(4-chlorophenyl)carbamoyl]-4-methyl-2-pyrazoline-4-carboxylate (Cl-pyrazoline) is one of the most potent compounds. Resolution of enantiomers is important to elucidate the more potent isomer in the insecticidal activity. A pair of enantiomers are also useful as a tool to study the mode of action, because they have same physical properties except for that they are nonsuperimposable mirror image. In the heterocyclic ring of Cl-pyrazoline, there is a chiral center giving a pair of enantiomers. We separated the enantiomers of the compound to compare their in vivo and in vitro activities. The S-isomer was about ten times more insecticidally active than the R-isomer when they were injected into the American cockroach. The factor of metabolic degradation was not much different from each other, so the difference of the isomers in the insecticidal potency seems to be a reflection of that at the target site. Voltage-dependent blocking effects of sodium channels have been found with this class of compounds by electrophysiological, sodium uptake, and binding assay methods. Another effect is block of voltage-sensitive calcium channels. Among the procedures to study the mode of action of this class of compounds with nerve preparations, we chose the ATP-dependent calcium uptake in rat brain synaptosomes. The calcium uptake increased up to about 20 min to reach a maximum level after the start of incubation with ATP. The maximum uptake was inhibited by Cl-pyrazoline and its optical isomers in a concentration-dependent manner. The S-isomer was substantially more active in the inhibitory effect than the R-isomer, suggesting that this effect may participate in the mode of action of this class of compounds.

O-12 ION CHANNELS AS TARGETS FOR INSECTICIDES Narahashi, Toshio Department of Molecular Pharmacology and Biological Chemistry, Northwestern University Medical School, Chicago, IL, USA

Among possible targets of insecticides, the voltage-gated sodium channel, the GABAA receptor chloride channel, the nicotinic acetylcholine receptor channel, and acetylcholinesterases have been identified as the major sites of action of pyrethroids/DDT, cyclodienes/ HCH, imidacloprid, and organophosphates/carbamates, respectively. Pyrethroid modifications of the sodium channel are characterized by slowing of both activation and inactivation gating kinetics that lead to prolonged openings of the sodium channel. This causes hyperexcitation of the nervous system. Only 1% of the sodium channel population needs to be modified to cause neuronal hyperactivity which leads to hyperexcitation of animals. This explains the high potency of the pyrethroids. The selective toxicity between mammals and insects is quantit- atively explicable largely in terms of the low sensitivity of mammalian sodium channels and negative temperature dependence. Therapeutic effects of vitamin E on paresthesia caused by pyrethroids are due to the selective block by vitamin E of pyrethroid-modified sodium channels. Differential sensitivities of mammalian and insect sodium channels to pyrethroids may be due to their differences in amino acid compositions, and newer selective insecticides could be created based on their specific affinity for the insect sodium channel. Cyclodienes and HCH were shown to block the GABAA receptor through binding and Cl- flux experiments, and we have found by patch clamp studies that these insecticides exert a dual action, stimulation and inhibition. Modulation of the neuronal nicotinic acetylcholine receptor channel by imidacloprid has recently been characterized by patch clamp experiments.

O-13 INSECT IONOTROPIC RECEPTORS AS TARGETS FOR INSECTICIDES Sattelle, D.B., Rauch, J.J.* The Babraham Institute Laboratory of Molecular Signalling, Department of Zoology, University of Cambridge, Cambridge, UK, * Du Pont Agricultural Products, Stine-Haskell Research Center, Newark DB, USA

Ionotropic receptors for the neurotransmitters acetylcholine (ACh) and g-aminobutyric acid (GABA) are members of the cys-loop receptor gene family. Such receptors are pentameric oligomers. Each polypeptide subunit has an N-terrninal extracellular region containing residues that contribute to the neurotransrnitter binding site. Also, each has four transmembrane regions (MI-M4), the second of which (M2) lines the integral ion channel. In the case of nicotinic acetylcholine receptor (nAChR) molecules, the ion channel is permeable to cations, whereas ionotropic GABA receptor (GABAR) molecules gate an anion channel. Functional expression of cloned insect ionotropic receptor subunits targeted by insecticides, together with parallel studies on native insect receptors is adding to our understanding of insecticide mode of action. As examples, recent work will be described on the interactions with Imidacloprid of insect nicotinic acetylcholine receptors (nAChRs), the known target of cartap and nicotinoids. In addition, the actions of Fipronil on ionotropic GABA receptors (GABARs), the known target of dieldrin, will be examined. Imidacloprid acts on pharmacologically distinct subtypes of native insect nAChRs. Recombinant nAChRs, formed by expressing cloned insect and vertebrate nAChR subunits in amphibian oocytes, have been used to investigate the role(s) of subunits determining the site and mode of action of Imidacloprid. Evidence for a partial agonist action is presented. A cloned insect GABAR subunit that forms robust functional ionotropic receptors when expressed in amphibian oocytes has also been studied. An A302S mutation that confers resistance to dieldrin and picrotoxinin also confers insensitivity to Fipronil, a potent blocker of the wild type receptor. Recently, we have developed stable insect cell lines expressing cloned insect ionotropic receptor subunits that are targeted by insecticides. Examples from studies with nAChrRs and GAßARs will be described.

O-14 STUDIES ON THE MECHANISM OF THE KDR-TYPE PYRETHROID RESISTANCE INVOLVING CHANGES IN THE FUNCTION OF SODIUM CHANNELS Matsumura, F., Miyazaki, M., Ohyama, K., Dunlap, Debra Y. Department of Environmental Toxicology, University of California, Davis, CA, USA

Using reverse transcription plymerase chanin reactions (RT PCR), the DNA sequence for the main membrane-spanning region (IS3 through IVS6) of the gene encoding the 8F-subunit of the para sodium channel of the German cockroach, Blattella germanica, has been determined. The overall structure of the open reading frame region of this B. Germanica gene is very similar to that of the para gene of Drosophila melaongaster, and that of the partially seqauenced para gene of Musca domestica. On the other hand, it is distinctly different from that of the DSC gene (Drosophila sodium channel). As a result of a side-by-side comparison of the para gene sequences of the susceptible CSMA strain and the Kdr resistance VT strain of B. Germanica, one mutation (TTG to TTC) at the approximate center of the IIS6 membrane-spanning segment was found to result in an amino acid change from L to F. While the functional meaning of this mutation for the operation of the para sodium channel remains to be studied, this reigon is very highly conserved among all sodium channels identified so far, and is one of the most hydrophobic areas of the entire 8F-subunit. For comparison, we have studied the same region of the para sodium channel of both kdr and susceptible SOB strain of the housefly, Musca domestica. We found the monologous type of mutation, CTT to TTT, resulting in the same amino acid alteration (L to F) at this site. The main question is how a mutuation at this site confers the sodium channel resistance to pyrethroids. IIS-6 is not even a part of the inner wall of the sodium channel itself. However, it is known that IIS-6 serves as an important connector for the II-III as well as the S5-S6 linker. The functional meaning of such an amino acid shift will be discussed.

O-15 THE PROBLEM OF DELAYED NEUROTOXICITY OF ORGANOPHOSPHORUS PESTICIDES (EXPERIMENTAL INVESTIGATION) V Kagan, Yu. S. Institute of Ecohygiene and Toxicology, Ministry of Health, Kiev, Ukraine

A number of organophosphorus compounds (OPC) are known to produce the delayed neurotoxic effect (DNE), which is manifested in the development of ataxia, paresis and paralyses of extremities during 2-4 weeks after contact. Dozens of thousands incidens of human neurophathy caused by triortocresylphosphate and some other OPC have been already described. Pathogenesis of these neuropathies has not been sufficiently studied yet. Aldrige, Jonhson, Lotti, Abou-Donia et al. in their studies came to the conclusion that neurotoxic esterase (NTE) is the enzyme target for OPC resulting in DNE. The experimental investigation of our laboratory using different OPC showed that fungicide aphos, O,O-diphenyl-1-acetoxy-2,2,2-trichlorethylphosphonate causes clinical manifestations of DNE in hens (I.I.Tkachenko et al., 1992). Aphos produces DNE at a wide dose level spectrum (25-3000 mg/kg) and inhibits NTE activity in hen and rat brain. Experiments in hens, guinea pigs showed that aphos in doses resulting in the development of clinical manifestations of the DNE, decreases the conduction velocity along peripheral nerves, produces demyelinization and degeneration in the sciatic nerve and spinal cord (I.I.Tkachenko et al.,1993). These and other investigations (Yu.S. Kagan et al.,1986,1995, N.V. Kokshareva et al.,1996) confirm the role of NTE as a target of OPC. We propose determination of NTE activity in vitro (without or after activation) and in vivo for screening of OPC on the DNE. It was shown that not only hens NTE, but also this enzyme from rat and guinea pig brain is sensitive to OPC, which have a property to cause DNE. Autoimmune processes play role in the development of neuropathy caused by OPC (P.G.Zminko et al.,1996). The supression of immune system by cyclophosphane diminishes the neurophathological lesions. There are some possibility for correction of the DNE by using of reactivators of ChE which penetrate in CNS (diethyxime) and promotors (before poisoning).

O-16 EXPERIMENTAL MODEL STUDIES OF PESTICIDE EXPOSURE Dési, I., Nagymajtényi, L., Schulz, H., Papp, A. Department of Public Health, Albert Szent-Györgyi Medical University, WHO Collaborating Centre for Chemical Safety, Szeged, Hungary

The working group investigated the neurotoxic effects of Dimethoate and Parathion-methyl respectively on the CNS and PNS after different treatment schedules exerted on macro and single unit cells level. 7,14, 28 mg/kg Dimethoate or 0,22, 0,44, 0,88 mg/kg Parathionmethyl was administered to different groups P.o.. daily in the following programs: Pregnancy variation(P): females 5th to l5th days of pregnancy; Pregnancy and lactation variation (P+L): females as above and during lactation for 4 weeks. Pregnancy+Lactation+Post weaning variation (P+L+P): as above plus the young male rats (F1 generation) further for 8 weeks. Neurotoxicological investigation was done on the F1 male rats at their age of 12 weeks. Spontaneous ECoG records were made on the anesthetized raats from the somatosensory, visual and auditory cortex. Cortical evoked potentials (EP) were recorded from the same areas subsequently. Conduction velocity and refractory periods of a peripheral nerve was investigated afterwards.

Results: spontaneous frequency grew significantly in the P + L+ P groups treatment of both pesticides, dose dependently. Latency time between stimuli and answers in EP showed the same characteristics. Conduction velocity of the tail nerve at the same groups diminished, refractory periods grew dose dependently. When studying cortical single unit activity after the i.p. admmistration of 1/5 LD50 of the OP-s, first transient increase of firing frequency was observed, later decrease of frequency occurred. There was no qualitative difference between the action of the OP-s. The activity of stimulus driven units showed a similar incrise-decrease sequence.

The work was supported by the Hungarian OTKA grant No: T016537

O-17 LONG-TERM NEUROTOXICITY OF CHLORPHENVINPHOS, AN ORGANO-PHOSPHOROUS PESTICIDE. DATA FROM BEHAVIOURAL AND ELECTRO-PHYSIOLOGICAL STUDIES ON RATS AND RABBITS Gralewicz, Slawomir, Socko, Renata Nofer Institute of Occupational Medicine, Lodz, Poland

Organophosphorous compounds (OP) constitute a large proportion of insecticides used all over the world. Their insecticide properties and acute toxicity in nontarget species derive from the inhibition of acetylcholinesterase (AChE) which results in the disturbances of the cholinergically mediated neurotransmission. However, as some literature data suggest, after either acute or prolonged exposure to OP subtle neurobehavioural impairments may persist long after normalization of AChE activity. The possibility that OP exposure may induce such long-term effects is nowadays a problem of great concern for the regulatory agencies. Here we present a review of studies from our laboratory aimed at detecting long-term neurobehavioural effects of exposure to chlorfenvinphos, an OP pesticide ((2-chloro-l-(2,4-dichlorophenyl) vinyl diethyl phosphate) - CVP)) in laboratory animals. In the United States all registered uses of CVP were canceled in 1991. In Poland, however, CVP is still manufactured (250 tons/year) and used for crop protection. In a series of experiments we have demonstrated that: a) in rabbits, two i.p. exposures to CVP at the same sublethal dose performed at three-month interval resulted in a similar inhibition of blood AChE activity but the effect of the second exposure on body temperature and hippocampal EEG was smaller and less consistent than that of the first one. This would indicate that some permanent changes within the CNS may occur even after a single exposure to CVP. b) in rats, under conditions of repeated i.p. exposure to CVP (one injection/day for ten days) at a symptomatic (3.0 mg/kg) dose inhibiting blood and brain AChE activity by about 80%, the tolerance to CVP, assessed on the basis of spontaneous locomotor behaviour, developed within four to five days. However, single exposure to CVP at a symptomatic (3.0 mg/kg) or subsymptomatic (1.0 mg/kg, less than 50% AChE inhibition) dose, or repeated exposure (one injection/day, for ten days) at subsymptomatic doses (1.0 mg/kg or 0.5 mg/kg) resulted in subtle changes in complex behaviours detectable after AChE activity in blood and in the brain had regained a normal level. The changes - neophobia in the open field, an increased and more persistent emotional response to a stressful stimulus, and increased EEG arousal response to an external pain signalling stimulus - suggest an increased reactivity of the system or systems responsible for the induction of fear; c) direct intrahypothalamic injections of CVP, unlike those of oxotremorine, a direct stimulant of cholinergic muscarinic receptors, did not induce overt changes in the animal (rabbit) behaviour and EEG. This would indicate that the changes in the CNS functions after CVP exposure may be the consequences of increased cholinergic activity due to AChE inhibition rather than to a direct stimulation of cholinergic muscarinic receptors by CVP. The above findings provide experimental evidence that health effects of exposure to OP, may persist after normalization of ChE activity in blood and in the brain.

O-18 ORGANOPHOSPHOROUS PESTICIDES EXPOSURE AND BEHAVIORAL CHANGES Hinkova, Liliana, Vergieva, T. National Center of Hygiene, Medical Ecology and Nutrition, Sofia, Bulgaria

The subject of the study were 45 men - agricultural workers (sprayers, mixer-loaders) applying mainly organophosphorous pesticides in an area with intensive plant protection practice and a control group (19 men) from the same area, having no occupational contact with pesticides. They were examined before and after the active spraying season. Neurobehavioral test battery and subjective symptoms questionnaire were used. Before the spraying season the examination did not detect any substantial differences between exposed and control groups. During the postseasonal investigation the neurobehavioral test revealed changes in psychomotor coordination (decrease in the number of correct dots in Aiming test and a lower performance in Digit Span test). The exposed subjects performed significantly worse than the controls. Positive correlation with education was established. The study has confirmed the applicability and feasibility of the implemented battery of tests.

O-19a ORGANOPHOSPHORUS PESTICIDES AND LONG-TERM HEALTH EFFECT Brown, I.****, Classen, W.**, Ivens, I.A.*, Krinke, G.J.**,Rieth, J.***, Rozenboom, W., Skripsky, Th.** ECETOC Belgium, *Bayer Inc. USA, **Novartis Inc. Switzerland, ***Rhône-Poulenc SA France, ****UBHT England

Organophosphorus pesticides (OP) have been known for decades. Owing to their potent biological effects, these agents are subject to thorough toxicological scrutiny aimed at the recognition of potential hazards and their risk management. In addition to "classical" hazards of acute toxicity and delayed neuropathy (OPIDN), more recently other specific types of OP neurotoxicity were reported, such as "intermediate syndrome", ocular "Saku disease" and "chronic syndrome" allegedly associated with long-term exposure. This prompted ECETOC to review existing epidemiological data and toxicological testing protocols used in development and registration of OPs, with respect to their sensitivity and reliability of detecting such effects. Epidemiological studies often lack sufficient determination of exact conditions and levels of exposure of the compounds involved. Data are frequently confounded by spontaneous occurrence of changes related to aging and underlying diseases. Most studies are retrospective, methods of examination are not standardized, frequently subjective, and rarely appropriately controlled. Chronic, low-level exposure may be associated with episodes of symptomatic intoxication. In these circumstances the signs, symptoms and sequelae may be wrongly attributed to low-level exposure. Similarly, acute effects induced by recent OP exposure may be mistaken as chronic effects of earlier exposure. These inaccuracies were perpetuated in many of the earlier reviews. Some epidemiological studies provide evidence of the absence of adverse effects in exposed, but well protected cohorts. Others report adverse effects, but at least in part, these effects differ with respect to the character of findings and are predominantly psychological, neurological or opththalmological. Animal experiments aimed at further characterizing neurotoxicity induced by specific OPs confirm acute and delayed effects on cognitive functions but have not demonstrated the alleged effects of prolonged, low-level exposure. Kinetic data demonstrate that inhibition of cholinesterase by multiple low doses increases only during the initial phase of exposure and thereafter reaches a steady state. Likewise, a weak inhibition of neuropathy target esterase has not been shown to be associated with the development of neuropathy. The reasons why chronic, low-level exposure could possibly culminate in chronic dysfunction have not been demonstrated. Several mechanisms have been proposed, such as changes in receptors or neurotransmission, potential non-cholinergic effects of cholinesterases or of OPs, or inhibition of other enzymes. However, these hypotheses are currently of academic interest and do not seem to be of practical importance for the understanding of OP neurotoxicity. This abstract represents preliminary results of the ECETOC Task Force which are subject to approval by the ECETOC Scientific Committee. The final report will be peer-reviewed by the Scientific Committee.

O-19b A CRITICAL REVIEW OF HAZARD IDENTIFICATION OF ORGANO-PHOSPHOROUS PESTICIDES Brown, I.****, Classen, W.**, Ivens, I.A.*, Krinke, G.J.**,Rieth, J.***, Rozenboom, W., Skripsky, Th.** ECETOC Belgium, *Bayer Inc. USA, **Novartis Inc. Switzerland, ***Rhône-Poulenc SA France, ****UBHT England

Based on the allegations that organophosphorus compounds (OPs) may induce prolonged or late effects, the adequacy of safety studies presently required for registration of these compounds is evaluated. A good hazard identification should correctly predict toxic effects which may occur in humans and provide a good basis for risk assessment. Data are presented showing that: 1) acute toxic effects are adequately predicted in a range of regulatory toxicity studies; 2) neuropathic OPs, inducing OPIDN, can be identified with sufficient accuracy in acute hen tests conducted according to the revised OECD or EPA guideline; 3) rodents and dogs are sensitive to OPIDN induced by repeated exposure indicating that neuropathy induced by prolonged exposure can be detected in standard subchronic or chronic toxicity studies; in the absence of guideline studies specifically aimed at chronic effects induced by acute or prolonged exposure to OPs, chronic effects induced by prolonged exposure are expected to show up also in subchronic special neurotoxicity and chronic standard toxicity studies.

The parameters observed in these tests appear to be sufficiently sensitive and reliable to detect neurotoxic materials given the limitations of interspecies extrapolation The potential improvement of the hazard identification process resulting from inclusion of additional tests or endpoints into neurotoxicity studies with OPs cannot be estimated yet. The presently valid guidelines can be expected to contain all essential elements needed for the evaluation of the neurotoxicity and provide sufficient flexibility for better characterisation of the observed effects when required. It is recommended that appropriate epidemiological surveillance is continued with observation of exposed populations to conclusively identify the presence or absence of chronic effects.

This abstract represents preliminary results of the ECETOC Task Force which are subject to approval by the ECETOC Scientific Committee. The final report will be peer-reviewed by the Scientific Committee.

O-20 DEVELOPMENTAL NEUROTOXICAL EFFECTS OF ENVIRONMENTAL POLLUTANTS (HEAVY METALS + ORGANOPHOSPHATES) IN ANIMAL EXPERIMENTS Nagymajtényi, L., Schulz, H., Papp, A., Dési, I. Department of Public Health, Albert Szent-Györgyi Medical University and WHO Collaborating Centre for Chemical Safety,Szeged, Hungary

In our previous studies it was stated that the pre- and/or postnatal treatment of low- level inorganic heavy metal compounds (C4H6O4Pb·x 3H2O, HgCl2, CdCl2) or organo-phosphorous pesticides (dimethoate, dichlorvos, parathion-methyl) dose and treatment-variation dependently changed the spontaneous and evoked bioelectric functions of rat's brain. In the recent experiment we investigated the combined effects of the low-level, pre- and/or postnatal, parallellous treatment of rats with inorganic heavy metal salts (C4H6O4Pb·x 3H2O, HgCl2, CdCl2) and dimethoate. Wistar rats were orally treated by gavage by 80.0, 160.0, and 320,0 mg/kg lead (in form of C4H6O4Pb·x 3H2O); 0.4, 0.8, and 1.6 mg/kg mercury (in form of HgCl2); 3.5, 7.0, and 14.0 mg/kg cadmium (in form of CdCl2), and 5.0; 10.0 and 20.0 mg/kg dimethoate. The above mentioned doses were given the female rats in 5-15 days of pregnancy (P variation), or in 5-15 days of pregnancy + for 4 weeks of lactation (P+L variation), or in 5-15 days of pregnancy + 4 weeks of lactation + 8 weeks postweaning (P+L+P variation). The electrophysiological parameters (ECoG, cortical evoked potentials, conduction velocity and refractory periods of peripheral nerve) of male rats of the differently treated groups were investigated at the age of 12 weeks. It was found that the metal + dimethoate combinations dose- and treatment duration-dependently altered both the spontaneous and evoked electrophysiological functions (changed ECoG frequencies, lengthened latencies and durations of evoked potentials, lower conduction velocity of peripheral nerve, etc). The changes caused by the combined treatment of metals and dimethoate were more expressed than in case of single administration of the mentioned compounds. The data showed that the low-level, pre- and postnatal combined exposure of certain metals and an organophosphate considerably affected the bioelectrical function of the nervous system without any signs of intoxication. Our results emphasize the real risk of the continuous (including pregnancy and lactation) low-level metal + organophosphate intake. The work was supported by the Hungarian OTKA grant No T016735

O-21 EFFECT OF A PESTICIDE (DELTAMETHRINE) ON THE INTESTINAL NERVOUS SYSTEM AND LIVER OF BALATON LAKE FISH Láng, G., Kufcsák, O., Ferenczi, J., Nemcsók, J., Knyihár-Csillik, E.*, Csillik, B. Bay Zoltán Institute for Biotechnology, *Department of Neurology, Albert Szent-Györgyi Medical University, Szeged, Hungary

Deltamethrin (DM), a synthetic pesticide used for extermination of mosquitoes on the shores of lake Balaton, has been found to induce severe impairment of the intestinal nervous system of several Balaton fish. Gut and liver of eel and carp have been studied by light- and electron microscopic, histochemical, enzyme histochemical and immunocytochemical techniques. Histochemical results are supported by biochemical measurements. As a toxic effect of DM, acetylcholinesterase (AChE) activity of the myenteric plexus is reduced, that of the nerve fibers innervating the muscular layers proper virtually disappears. The alterations are most marked in the foregut while in the mid-gut, the effect of DM is moderate. In the hind-gut, the effect is less marked than in the foregut but stronger than in the midgut. Even more striking is the decrease of the enzyme cholin acetyltranferase (ChAT) which is present, under normal conditions, in nerve cells of the myenteric plexus. In contrast, the amount of calcitonin gen-related peptide (CGRP) is conspicuously increased one and two days after DM treatment of eel; longer exposure, however, or higher doses, result in decreased CGRP both in the myenteric plexus and in nerve fibers innervating the muscle layers. In the carp, the alterations appear on a higher level since, under normal conditions, AChE activity and ChAT immunoreactivity of the carp gut is stronger than in the eel. It is assumed that the increased CGRP content of the gut as an effect of DM might reflect direct effect of the toxin on the genome, in manner similar to colchi which has been shown to increase CGRP mRNA levels. - The liver is a very sensitive against the toxic effect of DM, both in eel and in carp. Alterations ensue in increasing order with the time of exposure and the concentration of DM. Light microscopically, the toxic alterations result in a honeycomb-like appearance of hepatocytes in semi-thin sections; according to histochemical studies, this due to the accumulation of lipid droplets in the cells. Under the electron microscope, deterioration of the endoplasmic reticulum, shrinkage of mitochondria and the appearence of lipid-containing vacuoles have been noted. Also epithelial cells of intrahepatic bile ducts exhibit toxic alterations after 3 day exposure to DM. Our studies prove that DM induces severe irreversible damage to the intestinal nervous system and liver of Balaton fish. On the basis of the toxicity of DM, use of biological agents instead of DM as mosquito-killing pesticides is recommended.

O-22 BEHAVIORAL EFFECTS OF OCCUPATIONAL EXPOSURE TO ORGANO-PHOSPHOROUS PESTICIDES IN FEMALE GREENHOUSE PLANTING WORKERS Bazylewicz-Walczak, Barbara The Nofer Institute of Occupational Medicine, Lodz, Poland

The study covered 52 women employed in gardening enterprises. 26 workers involved in planting were occupationally exposed to several organophosphates in greenhouses. There were 26 non-exposed to neurotoxic chemicals women in the control group. The groups were similar in terms of age, place of habitation, workload, stimulants and drugs taken. The exposure measurements were performed during intensive using of pesticides from March to June. Environmental study included air pollution, contamination of skin and clothes, and work timing. The total exposure magnitude in the planting group reached a low level. Psychological examination was conducted twice: before and after spraying season. The Neurobehavioral Core Test Battery recommended by WHO was used. The results of psychological tests indicated no negative effects of exposure after a single spraying season. Analysis of the results, however, proved the existence of certain permanent effects of chronic occupational exposure. The exposed female workers were characterized by a slowing down of perceptual motor functions, weaker auditory memory and longer reaction time. In addition, increased tension, greater depression and fatigue, more frequent symptoms of CNS disturbances have been observed in the exposed women compared to the controls.

O-23 EPIDEMOLOGICAL EVIDENCE FOR INCREASED LEUKAEMIA RISK FROM PESTICIDE USE IN THE INDOOR ENVIRONMENT Csicsaky, M., Butte, W.*, Walker, G.*, Greiser, E.**, Hoffmann, W.**, Hostrup, O.*** Niedersächsisches Sozialministerium, Hannover, *EUKOS GmbH, Plön, **Institut für Präventionforschung und Sozialmedizin, Bremen, ***Universität Oldenburg, Informationsstelle Pestizide und Gesundheit (IPuG), Oldenburg, Germany

In the vicinity of the former toxic waste dump site of Muenchehagen in northern Germany an elevated rate of leukaemia was suspected by a local practitioner. To look into the problem, the regional governments of North Rhine-Westfalia and Lower Saxony had an incidence study conducted covering all cases of leukaemia, lymphoma and myeloma which occurred in the years 1984-1990. This study confirmed the observation of the physician. As environmental contamination was restricted to the immediate neighborhood of the dump site, however, there was no reason to believe that emissions from the dump site could be the reason for the increased leukaemia risk. A case control study on 623 cases and 1186 matched controls was then launched to find out about the risk factors involved. While it is well known that pesticide spraying constitutes a cancer risk to farmers, quite unexpectedly, pesticide use in the home and the private garden turned out to be a risk factor for adult leukaemia in females (ALL: OR=5.2; CLL: OR=5.3) in the high exposure group (more than 2 preparations used). This result was doubted by part of the scientific community on the grounds that self assessment of the study population was the only basis of information on their actual exposure to pesticides. Therefore, another study was launched where vacuum cleaner dust samples from the homes of 44 cases and 335 controls were analysed for pesticide residues. The results of the case control study were confirmed, even though the level of significance was missed due to the small sample size.

O-24 GENETIC POLYMORPHISMS IN PARKINSON'S DISEASE Checkoway, H., Costa-Mallen, P., Farin, F.M., Costa, L.G. University of Washington, Department of Environmental Health, Seattle, Washington, USA

Parkinson's disease (PD) is a relatively common neurodegenerative disorder caused by an excessive loss of dopaminergic cells in the substantia nigra. Some familial aggregation has been noted, although most PD cases are sporadic and idiopathic. Recent theories of PD etiology emphasize the plausibility of interactions between endogenous or exogenous neurotoxicants that are selective for dopaminergic cells and genetically-determined susceptibility traits. MPTP, which is established as the experimental model of PD induction, provides a valuable framework for investigating genetic polymorphisms of relevant enzyme systems that either afford neuroprotection or activate proto-neurotoxicants. The cytochrome P450 enzyme, debrisoquine hydroxylase (CYP2D6), may detoxify MPTP, and accordingly genetic polymorphisms of CYP2D6 have been a research focus. Epidemiological findings from the early 1990s indicated that genotypic characterization of CYP2D6 poor metabolizer status was associated with marked increased PD risks; however, more recent results have been conflicting, with some showing no association. Type B monoamine oxidase (MAO-B) is a potentially relevant enzyme in PD because it plays an important role in dopamine catabolism, and also activates MPTP to the ultimate neurotoxic metabolite. Moreover, diminished MAO-B activity resulting from cigarette smoke exposure has been postulated as a possible explanation for the frequently observed inverse relation between smoking and PD. To date, no genetic polymorphisms of MAO-B have been linked consistently with PD susceptibility. Further exploration of mutant forms of MAO-B and the type A isozyme, MAO-A, is ongoing. There is very compelling evidence from human studies and from in vitro experiments with MPTP that mitochondrial dysfunction, particularly of Complex I, is a significant contributor to dopaminergic cell impairment and death. Identification of genetic polymorphisms of Complex I and other mitochondrial enzymes will undoubtedly stimulate applications to PD research. Ultimately, elucidation of the underlying relations between genetic polymorphisms and enzyme function will be needed before confident conclusions can be reached regarding PD susceptibility traits.

O-25 POLYMORPHISM OF APOLIPOPROTEIN E AND ALZHEIMER'S DISEASE Müller, W.E., Eckert, G.P., Eckert, A., Hartmann, H.* Department of Pharmacology, Biocenter, University of Frankfurt, Frankfurt, Germany, *Division of Neuroscience, Children's Hospital, Harvard Medical School, Boston, MA, USA

Human Apolipoprotein (Apo) E has an important role in cholesterol transport and plasma lipoprotein metabolism. It is synthesized primarily by the liver, but also by peripheral nerves, macrophages and brain astrocytes. Although not fully understood, Apo E appears to be involved in the development of the central nervous system (CNS) and its regeneration after injuries. In amyloid deposits associated with Down Syndrome, Creuzfeld-Jacob's and prion diseases Apo F is found. In Alzheimer's disease (AD) brains, APO E is immunochemical colocalized with senile plaques (SP), including vascular deposits, and in neurons containing neurofibrillary tangles (NFT). Both PS and NFT are hallmarks of the disease, presumably linked to the progression of AD and ultimately dementia. The gene for human Apo E is found on chromsosome 19 and exists in three common allelic forms (Apo E -e2, -e3, -e4). It is well established that the e4 allele of Apo E gene is overrepresented in groups of patients with late onset familial AD. However, it is not yet known by which mechanism Apo E affects AD pathology. It was demonstrated that Apo E binds beta-amyloid peptide (Ab) and enhances the peptide aggregation, possibly via a chaperon like mechanism. Thus it was assumed that Apo E genotype plays an important role in development of SP and Ab neurotoxicity. However, although Apo E e4 genotype is clearly linked with AD, there is neither a correlation between the genotype and SD number nor between genotype and cognitive decline in AD. Furthermore, Apo E failed to enhance the neuronal membrane disordering effect of Ab in vitro, one of the most promising mechanisms of Ab neurotoxicity. Therefore, other mechanisms seem to be crucial for the relationship between Apo E and AD. Findings that Apo E attenuates the toxicity of Ab in cell culture systems suggests a role in neuroprotection. This benefit action is possibly linked to the cholesterol homeostasis of nerve cells. In vitro, it was shown that cholesterol abolished the calcium amplifying effect of Ab in different viable cell systems. Moreover, membrane cholesterol attenuated in vitro the membrane disordering effect of Ab on AD brain membranes. It was speculated that the high binding capacity of Apo E e4 to Ab diminishes its curative function, further a lower beneficial activity of this gene product may lead to a stronger disease progression. Because Apo E acts at multiple points in the development and pathophysiology of the CNS, additional work is necessary to elucidate the mechanisms by which the Apo E genotype isoforms exert their effects in AD.

O-26 ALAD POLYMORPHISM AND LEAD NEUROTOXICITY Kelsey, K.T., Schwartz, B.S.*, Lee, Byung-Kook** Harvard School of Public Health, Boston, MA, USA, *Johns Hopkins School of Public Health, Baltimore, MD, USA, **Department of Preventive Medicine, Soonchunhyang University, Republic of Korea

Exposure to lead constitutes a serious occupational and environmental health problem. Several reports have indicated that a polymorphism in the aminolevulinic acid dehydratase gene (ALAD) is associated with higher blood lead levels in populations highly exposed to lead. We have previously confirmed this observation; showing a trend toward higher blood-lead levels in highly-exposed workers that is not apparent in environnmentally exposed individuals. This has led to the hypothesis that AL AD is a major lead-binding protein and that the variant form of the protein has different binding characteristics. In further study we have now shown that the ALAD gene polymorphism modifies four--hour urinary lead excretion after oral administration of dimercaptosuccinic acid. We have also found ALAD genotype to be associated with different profiles of hemoglobin subtype in exposed workers, perhaps indicating that lead binding affects red blood-cell maturation or production in workers with different genotypes. Finally, we have shown that ALAD genotype mediates plasma levels of the neurotoxin 5-aminolevulinic acid (ALA) in highly lead-exposed workers. This work indicates that this polymorphism may be very important in determining susceptibility to the toxic effects of lead by modification of its pharmacokinetics. It may also be associated with susceptibility to some of the adverse neurological effects of lead poisoning, if ALAD contributes to the induction of lead-related central nervous system decrements. Supported by NIEHS ES-07198, NIEHS P42 ES -05947, NIEHS ES-00002

O-27 GENETICALLY DETERMINED VARIABILITY OF RESISTANCE TO ORGANO-PHOSPHORUS INSECTIDES AND NERVE AGENTS: THE HUMAN PON1 POLYMORPHISM Furlong, C.E., Li, W.-F., Costa, L.G., Richter, R.J., Shih, D.*, Lusis, A.J.* Department of Medicine and Genetics, University of Washington, *University of California at Los Angeles, USA

Several organophosphorus insecticides and nerve agents are detoxified through the cytochrome P450/paraoxonase (PON1) pathway. PON1 is an HDL-associated enzyme of 355 amino acids in humans. The PON1 Arg 192 isoform hydrolyzes paraoxon rapidly while the Gln192 isoform hydrolyzes this compound slowly. Both isoforms hydrolyze phenylacetate and chlorpyrifos oxon at approximately the same rate. We recently found that the effect of this polymorphism is dramatically reversed for sarin hydrolysis. The PON1 Arg 192 isoform has virtually no sarinase activity while the Gln 192 isoform has substantial activity. The Gln 192 isofom also hydrolyzes diazoxon and soman faster than the Arg 192 isoform. In addition to the large differences in rates of hydrolysis observed for some OP substrates by the two PON1 isoforms, there is also a large variability in serum PON1 concentrations that is stable over time between individuals. Thus, two factors govern the PON1 status of a given individual, the PON1 genotype as well as the amount of protein expressed from each allele. A two-dimensional enzyme analysis provides an excellent assessment of an individual's PON1 status , i.e. the position 192 genotype as well as phenotype, or level of serum PON1 (Nature Genet. 14:334-336). Do these interindividual differences in rates of substrate hydrolysis by PON1 reflect an individual's sensitivity or resistance to OP compounds processed through the P450/PON1 pathway? Injection of purified PON1 into mice clearly demonstrates the protective effect of having high serum levels of PON1 against toxicity by chlorpyrifos oxon or chlorpyrifos. Preliminary experiments with PON1 knockout mice, on the other hand, clearly demonstrate that low PON1 levels result in dramatically increased sensitivity to chlorpyrifos oxon. Supported by NIH Grant ES 05194.

O-28 METALOTHIONEINS AS POTENTIAL NEUROPROTECTANTS Aschner, M. Department of Physiology and Pharmacology, Bowman Gray School of Medicine of Wake Forest University, Winston-Salem, NC, USA

It is well established that astrocytes play a pivotal role in the etiology of mercury-induced neurotoxicity. Mercury preferentially accumulates in astrocytes, both in humans and non-human primates. Both organic (methylmercury; CH3Hg+) and inorganic mercurials (mercuric; Hg2+) inhibit glutamate and aspartate uptake in astrocytes. Furthermore, exposure of astrocytes to mercurials is associated with astrocytic swelling both in vivo and in vitro, inhibition of the ensuing regulatory volume decrease (RVD), and increased release of endogenous excitatory amino acids, such as glutamate and aspartate. That metallothioneins (MT) may attenuate neurotoxicity is consistent with recent observations that MT induction both in peripheral tissues and more recently in the CNS afford cellular protection against heavy metal cytotoxicity. For example, MT transcription is induced by cadmium and enhanced MT gene expression is associated with protection from cadmium-induced genotoxicity in cultured rat liver cells, and lead-induced toxicity in Chinese hamster ovary (CHO) cells. Similarly, targeted disruption of MT-I and MT-II genes increases sensitivity to cadmium. Cultured cells selected for resistance to cadmium have elevated MT levels; embryonic cells isolated from a transgenic mouse deficient in MT-I and MT-II genes retain their enhanced sensitivity to cadmium, and finally, cultured astrocytes induced to overexpress MT-I and MT-II levels by pretreatment with cadmium are resistant to the cytotoxic effects of the organometal, methylmercury. Studies from our laboratory suggest that pre-exposure of astrocytes to cadmium completely reverses the inhibitory effect of methylmercury on [3H]-D-aspartate uptake which occurs in methylmercury-treated astrocytes with constitutive MT levels. Increased MT expression may thus represent a generalized response to heavy metal exposure, protecting astrocytes, and perhaps also indirectly, juxtaposed neurons. It is noteworthy that in cells exposed to highly toxic metals, such as methylmercury and cadmium, cytotoxicity appears at metal concentrations higher than those at which MT induction is saturated. This might reflect the toxicity exerted by free heavy metal ions in excess of the metal-binding capacity of induced MTs. Supported in part by grants from the National Institute of Environmental Health Services (ES 07331) and the Environmental Protection Agency (R-819210)].

O-29 MTF-1-INDEPENDENT TRANSCRIPTIONAL ACTIVATION OF MOUSE METALLOTHIONEIN I PROMOTER BY CADMIUM IN HUMAN NEUROBLASTOMA CELLS Johnson, J.A., Moehlenkamp, J.D., Chu, Wai H., Bittel, D.*, Andrews, Glen K. Departments of Pharmacology, Toxicology and Therapeutics, *Biochemistry and Molecular Biology, University of Kansas Medical Center, Kansas City KS, USA

Metallothioneins (MTs) are cysteine-rich proteins that bind metal ions with high affinity. In all cell types that have been examined, mouse MT-I (mMT-I) and MT-II are up-regulated by zinc (Zn), cadmium (Cd), as well as pro-oxidants (tert-butylhydroquione, tBHQ). The 5'-flanking region of the mMT-I gene contains multiple cis-acting elements that bind the trans-acting factors Sp1, MRE-binding transcription factor-1 (MTF-1) and upstream stimulatory factor (USF). There is also an antioxidant responsive element (ARE) which overlaps the USF binding site. The protein(s) that bind to the ARE have not been identified. Transcriptional activation of mMT-I has been extensively studied, however there is little data on regulation of the mMT-I promoter in neuronal cells. Thus, the purposes of this investigation were to determine if the mMT-I promoter is transcriptionally activated by metals and oxidative stress in human IMR-32 neuroblastoma cells, and to identify trans-acting factors involved in the up-regulation of mMT-I promoter. Truncations of the mMT-I promoter (-250mMT-I, -150mMT-I, and -42mMT-I) were inserted into a luciferase (luc) reporter construct. A -150_USF/ARE-luc construct was used to assess the role of the USF/ARE within the context of the intact promoter, whereas insertion of MREd5 (5 copies) and USF/ARE (1 copy) into a TATA-Inr minimal promoter-luciferase reporter was used to address the enhancer function of these elements. IMR-32 cells were cotransfected with the mMT-I luciferase constructs and a RSV-ß-galactosidase constitutive reporter, and treated with tBHQ (10 mM), Cd (0.5 mM), and Zn (100 mM). The -250 mMT-I-luc, -150 mMT-I-luc and -150 mMT-I_USF/ARE-luc were activated by Cd (7- to 12-fold). Remarkably, these same constructs were not activated by Zn or tBHQ. The -42 mMT-I-luc and USF/ARE-luc were not induced by treatment with tBHQ, Cd or Zn. Activation of the MREd5-luc construct followed the pattern seen with the complete promoter constructs: Cd induced the MREd5-luc (12- to 15-fold), whereas Zn and tBHQ were ineffective. Electrophoretic mobility shift assays suggest that a functional MTF-1 is not present in the IMR-32 cells. IMR-32 cellular lysates from control, tBHQ, Cd and Zn treated cells did not contain MRE-binding activity, and this activity was not activated by Zn in vitro. These data were contrasted with those from a mouse hepatoma cell line (Hepa-1). Hepa-1 cells treated with these compounds had MRE-binding activity and this activity is activated in vitro by Zn. The absence of MTF-1 could account for the lack of a Zn response. To test this hypothesis, cells were cotransfected with a mammalian expression vector for mouse MTF-1 (CMV-mMTF-1) and the mMT-I reporter constructs. These conditions regenerated a Zn-mediated induction for all the mMT-I reporter constructs and MREd5-luc. Notably, activation these constructs by Cd was unaffected by overexpression of mMTF-1. These data suggest that Cd activates the MRE by a MTF-1-independent mechanism in human IMR-32 neuroblastoma cells. Supported by ES 08089 & ES 05704

O-30 REGULATION OF BRAIN METALLOTHIONEINS Hidalgo, J. Department of Cellular Biology and Physiology, Unit of Animal Physiology, Autonomous University of Barcelona, Bellaterra, Barcelona, Spain

Metallothioneins (MTs) are a family of proteins characterized by their high heavy metal (Zn2+, Cu+) content and an unusual cysteine abundance. MT-I and MT-II are two MT isoforms which are expressed in most tissues including the brain, whereas MT-III (also called Growth Inhibitory Factor, GIF) and MT-IV are expressed predominantly in the CNS and in keratinizing epithelia, respectively. All MT isoforms have been implicated in disparate physiological functions such as Zn2+ and Cu2+ metabolism, protection against oxidative agents, or adaptation to stress, whereas MT-III has been involved additionally in neuromodulatory events and in the pathogenesis of Alzheimer disease. To gain insight into how MTs are regulated in the brain is therefore essential for elucidating the potential importance of these proteins for a normal brain physiology as well as in pathophysiological states. A major physiological inducer of MT-I+II isoforms in peripheral tissues such as the liver is acute stress. MT-I+II isoforms are also significantly induced in some brain areas (especially hypothalamus, cerebellum, medulla+pons and cortex) by a well established stress model, immobilization, suggesting that these isoforms may be involved in the physiological adaptation to stress. The effect of stress on MT-III mRNA levels differs between brain areas, paralleling in some cases MT-I+II changes whereas in others opposing trends between MT isoforms can be observed. The prototypical stress hormones, glucocorticoids and catecholamines, are involved in brain MT-I+II regulation in basal animals, but their role in MT-I+II response to stress is likely only for the former hormones but not all brain areas. Surprisingly, glucocorticoids appear to be also involved in MT-III regulation, as indicated by the effect of adrenalectomy in vivo and by results in primary cultures of neurons and astrocytes. Brain cytokines are also involved in brain MTs regulation. Thus, endotoxin upregulates brain MT-I+II and downregulates MT-III in both rats and mice. Transgenic mice expressing IL-6 under the regulatory control of the glial fibrillary acidic protein gene promoter show significantly increased MT-I+II protein levels in cerebellum, medulla+pons, hypothalamus and remaining brain, but not in hippocampus. Furthermore, IL-6 administered icv also upregulates MT-I+II, suggesting that IL-6 is a major regulator of brain MT-I+II and that these MT isoforms could be considered acute-phase proteins in the brain. Results with knockout mice for IL-6 further support a role of IL-6 in MT regulation, especially in the liver. The results therefore demonstrate that MTs are multiregulated proteins and that they could be considered members of the acute-phase response.

O-31 THE EFFECT OF TRIMETHYLTIN ON ACETYLCHOLINE RELEASE IN THE GUINEA-PIG TRACHEA Aas, P., Pagenhart, A., Eriksen, S., Kolderup, J., Fonnum, F. Norwegian Defence Research Establishment, Division for Environmental Toxicology, Kjeller, Norway

The purpose of the present work was to characterise the effects of trimethyltin on the release of acetylcholine from parasympathetic nerves. Trimethyltin (3.0x10-3M) significantly enhanced in a reversible manner the high K+ (75 mM) evoked release of endogenous acetylcholine and [3H]acetylcholine. The evoked release of endogenous acetylcholine and [3H]acetylcholine was released from a pool of acetylcholine being independent of extraneuronal Ca2+ in the presence, but not in the absence of trimethyltin. The effect of trimethyltin on the release was not inhibited by low Ca2+ or by Ca2+ channel blockers (verapamil, flunarizine, w-conotoxin GVIA and w-agatoxin). The present results also demonstrate that trimethyltin induces emptying of a non-vesicular, probably a cytoplasmic storage pool of acetylcholine, since AH5183 (2.0x10-5 M) and a-latrotoxin (l.0xl0-8 M) had no inhibitory effects on the release of [3H]acetylcholine evoked by trimethyltin. The release of [3H]acetylcholine was moreover enhanced by trimethyltin when the vesicular uptake of [3H] acetylcholine was inhibited by AH5183, probably as a result of a higher cytoplasmic concentration of [3H] acetylcholine. Trimethyltin also reduced the neuronal uptake of [3H]choline and this was probably due to a depolarising effect of trimethyltin on the cholinergic nerve terminals. A similar depolarisation induced by trimethyltin was observed during patch-clamping of GH4 C1 neuronal cells. Furthermore, the reduced electrical field-induced contraction of the tracheal muscle and the subsequent increase in the basal smooth muscle tension that was observed by addition of trimethyltin was activity-dependent, and was most probably due to emptying of a nervous non-vesicular storage pool of acetylcholine, followed by rapid hydrolysis of acetylcholine by acetyl- and pseudocholinesterases.

O-32 EVIDENCE FROM A POPULATION-BASED STUDY OF NEUROBEHAVIORAL DEFICITS IN US PERSIAN GULF VETERANS WITH UNEXPLAINED SYMPTOMS Anger, W.K., Storzbach, D.*, Binder, L.M.*, Campbell, K.A.*, Rohlman, Diane S., McCauley, Linda, Kovera, C.A., Davis, Kelly L. Center for Research on Occupational and Environmental Toxicology Oregon Health Sciences University Portland, OR., and **Portland Environmental Hazards Research Center (PEHRC) Portland VA Medical Center, USA

Randomly-selected Northwest US veterans of the 1991 Persian Gulf War were sent questionnaires regarding nationally-recognized symptom complaints related to Persian Gulf service and possible chemical exposures while there. Following extensive interviews and physician exams to exclude persons who had medical explanations of their symptoms, veterans with persistent cognitive/psychological, gastrointestinal, dermatologic, fatigue or muscle and joint pain symptoms beginning during or after Gulf service constituted approximately 2/3 of the respondents ("cases"), while 1/3 reported no such symptoms ("controls") in this ongoing study. Cases studied through 1996 differed substantially and significantly from Controls on a broad range of psychological tests indicative of increased distress, including measures of posttraumatic stress disorder, health symptoms, personality, and symptoms of psychopathology. In addition, a subgroup of Cases was identified with objective deficits, compared to Controls, on neurobehavioral tests of memory, attention, and response speed.

O-33 DEVELOPMENTAL NEUROTOXICITY STUDY: EVALUATION OF BEHAVIOURAL METHODS WITH METHYLAZOXYMETHANOL AS POSITIVE CONTROL AGENT Hass, U., Lund, S.P., Simonsen, L. Department of Toxicology and Biology, National Institute of Occupational Health, Copenhagen, Denmark

In recent years, increased attention has focused on improving and standardizing methods for hazard identification of developmental neurotoxicants. The OECD Test Guideline Program for the testing of chemicals has scheduled to develop a guideline for Developmental Neurotoxicity Study. The functional endpoints to be included are reflex ontogeny, motor activity, motor function, sensory function and learning and memory function. The neurobehavioral test battery used in our laboratory includes assessment of motor activity in an automated Open Field, and investigations of learning and memory abilities in the Morris water maze. In order to evaluate these methods, pregnant rats (Mol:WIST) were dosed i.p. on gestation day 15 with 30 mg/kg methylazoxymethanol (MAM). MAM induces microencephaly in rats and studies of MAM have consistently reported hyperactivity, and learning deficits in mazes. Microcephaly in the offspring was verified by a marked reduction in brain weight. In Open Field, the MAM-exposed offspring were hyperactive compared to controls. The investigations of learning and memory functions clearly demonstrated effects of prenatal MAM on learning in the Morris water maze. The impairment seemed not to be related to poorer swimming capabilities, because the path lengths were longer in proportion to the increased latencies. These findings support the view that hyperactivity and learning deficits can be detected reliably in respectively Open Field and the Morris water maze and therefore suggest that these behavioral test methods can be useful in a neurobehavioral test battery for identification of developmental neurotoxicants.

O-34 INTERVENTIONS DURING INDIVIDUAL DEVELOPMENT OF RATS AFFECT THE BEHAVIOUR IN ADULTHOOD: A THREE-GENERATION STUDY Schulz, H., Nagymajtényi, L., Dési, I. Department of Public Health, Albert Szent-Györgyi Medical University, Szeged, Hungary

The investigated substances (lead, mercury and cadmium) have a different dose- and exposure-time dependent action profile on behavioural pattern of adult male Wistar rats, like spontaneous exploratory behaviour of an open field box, when the individuals exposed during development. There are no compensatory or adaptive mechanisms to eliminate xenobiotical effects throughout the generations. In contrast, it seems they are strengthened. The developing rats are extremely sensitive to neuro-behavioural damaging by heavy metals during pregnancy whereas further exposure mostly did not intensify the effects. The tested substances exhibit beside their behavioural effects also teratogenic effects on different organs of the adults which interfere directly or indirectly with the well-being of the individuals leading to neuro-physiological malformations and inadequate and/or inadaptive information processing in the central nervous system. The question of interactions between the existing xenobiotics in the biosphere on individuals information processing in the CNS is still open for further investigations. Supported by the Hungarian OTKA grant No.T016735.

O-35 LEARNING AND MEMORY DEFICITS INDUCED BY PRENATAL ALUMINIUM EXPOSURE IN RAT PUPS Lehotzky, Kornélia, Gonda, Zsuzsanna*, Tátrai, Erzsébet Department of Toxicology, National Institute of Occupational Health, *Department of Comparative Physiology, Faculty of Science, Loránd Eötvös University, Budapest, Hungary

Prenatal administration of neurotoxic agents has been shown to lead to subtle neurobehavioural impairments manifested by developmental delays and subsequent learning deficits - so called minimal brain dysfunction - without morphological abnormalities. Behavioural teratogenic effects have been demonstrated following prenatal exposure to different compounds of lead, mercury, tin and cadmium. Aluminium (Al) has been implicated as a neurotoxic agent, dementia has been correlated to elevated Al levels in Alzheimer's disease and has been related to impaired motor coordination and to a number of cognitive deficits. To determine the neurotoxicity of Al lactate exposure in rat pups, postnatal behavioural effects of 0, 2.5, 5 and 10 mg/kg daily s.c. treatment during gestational days 7th-l5th were investigated by using our test battery. The main sign of neurotoxicity was diminished performance and lengthened latency in an avoidance responding task in all treated groups, during acquisition, extinction and reconditioning as well. Neurohistology of pups treated prenatally with top dose showed no alterations in the cerebellar Purkinje cells or in the hippocampus. Lack of neurofibrillary degeneration (tangles) and axonal swelling also strengthened the species differences. Our findings support that Al-lactate is a developmental neurotoxicant and might induce impairment in cognitive and associative functions in a very low dose.

O-36 EFFECTS OF LOW LEVEL LEAD EXPOSURE DURING DIFFERENT PERIODS OF BRAIN DEVELOPMENT ON NR1 mRNA EXPRESSION IN RAT HIPPOCAMPUS Stoltenburg-Didinger, G., Peters, B. Institute of Neuropathology, Free University Berlin, Germany

Low level lead exposure primarily affects the developing brain, causing behavioral and cognitive alterations in humans and animals. Activation of the N-methyl-D-aspartate (NMDA) receptor in the hippocampus is essential for long-term potentiation (LTP), a functional correlate of synaptic plasticity. In situ hybridization was used to compare NRl mRNA expression in the hippocampus of lead exposed Wistar rats. Animals were divided in four groups: a permanent group exposed during gestation, lactation and postweaning (EO-P70-250), a perinatal group exposed during gestation and postnatally until weaning (EO-P16), a postweaning exposed group (P16-P70-250) and a control group with no lead exposure. All exposed animals received 750 ppm lead acetate. In the perinatal group NRl mRNA expression was decreased in contrast to an increase in the postweaning group. The permanent group demonstrated NRl mRNA levels like controls. Differentiated neurons in the postweaningly exposed rats react with upregulation of NRl mRNA expression, indicating compensation capacity for functional deficits. Permanently exposed animals show insufficient compensatory upregulation, leading to NR1 mRNA levels identical to those of controls. Thus, low level lead exposure results in permanent downregulation of NRI mRNA expression in neurons with developmental exposure only.

O-37 SOMAN INHIBITS N-METHYL-D-ASPARTATE-STIMULATED [3H]NOREPINEPHRINE RELEASE FROM RAT CORTICAL SLICES Tang, H.W., Cassel, G. Department of Biomedicine, Division of NBC Defense, National Defence Research Establishment, Umea, Sweden

Soman, an irreversible cholinesterase inhibitor, is known to produce convulsions, seizures and subsequent brain damage. Although the increases of acetylcholine and extracellular glutamate levels in rat hippocampus were reported by microdialysis during seizures induced by soman, the effects of soman on the release of neurotransmitters and its mechanisms have not been properly investigated. This study was undertaken to observe the effect of soman on N-methyl-D-aspartate (NMDA)-stimulated [3H]norepinephrine (NE) release from rat cortical slices. NMDA increased concentration-dependently the [3H]NE release, which was inhibited by soman, but potentiated by carbachol. Soman decreased the release of [3H]NE evoked by NMDA in a concentration-dependent manner. Atropine attenuated the increased release of [3H]NE induced by NMDA in the presence of carbachol or acetylcholine, but caused no changes of release in the presence of soman. Soman also inhibited significantly the elevated K+-induced release of [3H]NE. These results suggested that non-cholinergic effects may be involved in neurotoxicity induced by soman. Soman inhibits NMDA-activated [3H]NE release in cortical slices, probably due to both a direct effect at the NMDA-ion channel complex and by interfering with depolarization-evoked stimulus-secretion coupling. The exact mechanisms need to be further studied.

O-38 NEURAL LESIONING WITH RIBOSOME-INACTIVATING TOXINS Wiley, R.G. Vanderbilt University and VAMC, Nashville, TN, USA

The use of targeted cytotoxins to make highly selective neural lesions began with ricin. Ricin, the toxic lectin from castor beans, applied to peripheral nerves produce anatomically restricted destruction of motor and sensory neurons projecting through the treated nerve. The lesion results from endocytosis of ricin, retrograde axonal transport to the cell body and irreversible inhibition of protein synthesis. The role of nervous effects in fatal systemic ricin poisoning is unclear. When applied to the cervical vagus, vagal neurons stop functioning between 36 and 48 hours and disintegrate over the next 5-7 days. Other toxic lectins with similar effects on peripheral nerves include abrin, viscumin, volkensin and modeccin. Volkensin and modeccin differ from ricin by being active within the central nervous system. Both volkensin and modeccin have been used to make anatomically selective CNS lesions based on endocytosis and retrograde transport to cell bodies. Examples of uses for these suicide transport agents have included determining the cellular localization of neurotransmitter receptors, analysis of the mononuclear response to neuronal degeneration, somatosensory plasticity after peripheral nerve ablation and transsynaptic remodeling after selective ablation of a particular type of neuron. None of the toxic lectins is usefully selective for particular types of neurons. Immunotoxins harness the selectivity of monoclonal antibodies to control targeting of toxin to particular types of neurons. The first anti-neuronal immunotoxin, OX7-saporin, was made as a "proof of concept." This immunotoxin is effective in killing any neuron, peripheral or central, by local application or axonal transport, thus it is comparable to, but safer, than volkensin and modeccin. A unique feature of intraventricular OX7-saporin is the preferential destruction of cerebellar Purkinje neurons. The first selective anti-neuronal immunotoxin was 192-saporin which is directed at neurons expressing the low affinity NGF receptor, p75NGFr. When injected systemically, this immunotoxin kills postganglionic sympathetic and some dorsal root ganglion neurons. Injected intraventricularly, 192-saporin, kills cholinergic neurons of the basal forebrain and cerebellar Purkinje neurons. Anti-D H-saporin, is directed at dopamine -hydroxylase and therefore is selective for adrenergic and noradrenergic neurons, centrally or peripherally. Recently, anti-DAT-saporin, an immunotoxin directed at the dopamine transporter (DAT), has shown promise for selectively destroying midbrain dopaminergic neurons. All of these immunotoxins have been and are being used to model neurodegenerative disorders such as Purkinje cell degeneration, Alzheimer's and Parkinson's diseases. The newest additions to the armormentarium of targeted anti-neuronal toxins are neuropeptide- saporin conjugates, SP-saporin and SSP-saporin. These toxic conjugates consist of substance P (SP) or a stable analog (SSP) coupled to saporin and therefore, are selectively toxic for neurons expressing the neurokinin-1 receptor (NK-1R). When injected into the striatum, SP-saporin selectively destroys striatal interneurons that express NK-1R. When injected intrathecally into the lumbar spinal CSF, SSP-saporin produces loss of NK-1R expressing neurons from the lumbosacral spinal cord and decreased pain sensitivity in the tail and hindlimbs of rats. Thus, SP-saporin and SSP-saporin promise to be particularly useful in studies of the neurobiology of pain and possibly for treatment of chronic intractable pain. Other neuropeptides, such as somatostatin and neurotensin, also are likely to selectively targert saporin. Targeted anti-neuronal cytotoxins have already proven useful in a number of experimental strategies, for modelling neurodegenerative disorders and may have therapeutic potential in pain.

O-39 MODELING NEUROLOGICAL DISORDERS WITH IMMUNOTOXINS: THE 192-IgG-SAPORIN MODEL OF ALZHEIMER'S DISEASE Walsh, Thomas J. Department of Psychology, Rutgers University, New Brunswick, NJ, USA

Immunotoxins are conjugates of a monoclonal antibody targeting a specific antigen, combined with a ribosome-inactivating protein (RIP). An immunotoxin's antibody component recognizes and attaches to a highly specific membrane-associated antigen. The antibody and its coupled RIP is internalized by receptor-mediated endocytosis and is then transported to the cell body where it irreversibly halts protein synthesis, resulting in cell death. Since immunotoxins recognize and destroy only antibody-targeted cells it is possible to create highly selective lesions which can mimic neurodegenerative disorders and/or address fundamental neurobiological questions. Discussion will focus on the history of immunotoxins, their potential contributions to basic and clinical neuroscience, and the current data describing the neurobiological and behavioral effects induced by 192-IgG-saporin. 192-IgG-saporin is an immunotoxin that combines the 192 IgG monoclonal antibody to the p75 low affinity neurotrophin receptor with saporin, a potent RIP derived from Saponaria officinalis. The immunotoxin targets the p75 receptor localized on cholinergic nerve terminals in neocortex and hippocampus and on cholinergic cell bodies in the basal forebrain. This compound is an important new tool to (i) explore the biological and behavioral properties of the cholinergic basal forebrain (CBF), (ii) examine strategies to limit, attenuate, or reverse the functional consequences of damage to the CBF, (iii) determine the plasticity of this and interacting brain systems following selective insult, and finally (iv) model the functional deficits which occur following damage to the CBF in neurological disorders such as Alzheimer's disease.

O-40 NEURAL TRANSPLANTATION IN THE 192 IgG-SAPORIN DEPLETED RAT BRAIN Leanza, G., Nilsson, O.G.*, Nikkah, G.*, Martinez-Serrano, A.*, Wiley, R.G.**, Björklund, A.* Dept. of Physiological Sciences, University of Catania, Italy, *Wallenberg Neuroscience Center, University of Lund, Sweden, **Neurology Service, VAMC and Vanderbilt University, Nashville, TN, USA

The recent introduction of a powerful immunotoxin, 192 IgG-saporin, able to selectively target and efficiently lesion cholinergic neurons in the basal forebrain has provided an opportunity not previously available to address the issue of a relationship between cholinergic hypofunction and cognitive decline originally stated in the cholinergic hypothesis of geriatric memory dysfunction. In the present series of experiments, the 192 IgG-saporin lesion model has been employed in neonatal and adult rats to investigate respectively: (a) the functional integration and reinnervation capacity of embryonic basal forebrain cholinergic neurons following homotopic placement into the immunotoxin-depleted septum; and (b) the ability of such grafts, combinedly transplanted to the depleted hippocampus and cortex, to ameliorate the severe learning and memory deficits induced by the immunotoxin intraventricularly. The results show: in neonatal hosts, that homotopically implanted cholinergic neurons survive and integrate well into the developing septal area, extending axons along major myelinated pathways that reinnervate the appropriate target territories with remarkable accuracy; in adult hosts, that cholinergic-rich grafts, but not control grafts of fetal cerebellar tissue can efficiently reverse the spatial navigation and short-term memory deficits induced by the lesion, and that such graft-promoted recovery correlates highly with the indices of cholinergic fiber density in the reinnervated terminal territories. Thus, the 192 IgG-saporin lesion model appears to be highy suitable for experimental studies aimed at elucidating the role of the basal forebrain cholinergic system in cognition, also providing a valuable tool to explore the possibility of functional reconstruction of the severed ascending cholinergic pathways, following neural transplantation in the rat brain.

O-41 THE EFFECTS OF ETHYLBENZENE EXPOSURE ON HEARING IN RATS Cappaert, Natalie L.M., Muijser, H.*, Klis, S.F.L., Kulig, B.M.*, Smoorenburg, G.F. Hearing Research Laboratories, Dept. of Otorhinolaryngology, Utrecht University, The Netherlands, *Division Toxicology, TNO Toxicology, Zeist, The Netherlands

Exposure to organic solvents has been shown to be ototoxic in animals and there is evidence that these solvents could cause hearing loss in humans. In this study, the effects of inhalation of the possibly ototoxic solvent Ethylbenzene were evaluated using three complementary techniques: 1) measurement of the acoustic startle response (ASR), 2) electrocochleography and 3) histological examination of the cochleae. Rats (Wag/Rij, 200 gr) were exposed to either Ethylbenzene by whole body inhalation (800 ppm, 8 hours/day for 5 days) or to control conditions. The ASR was performed 1 week before and 1 and 4 weeks after exposure. After exposure the auditory thresholds increased significantly for 4, 8, 12, 16, 20 and 24 kHz stimuli. Electrocochleography was performed between 8 and 11 weeks after the exposure to organic solvents. Preliminary results showed smaller threshold shifts for the compound action potential at all frequencies (0.5 - 24 kHz) in comparison with ASR. Histological examination of the cochleae, which is presently in progress, will provide more insight as to the origin of the Ethylbenzene evoked ASR threshold shifts.

O-42 NEUROTOXICITY ASSOCIATED WITH EATING FISH FROM THEST-LAWRENCE RIVER Mergler, Donna, Bélanger, S., Larribe, F., Panisset, M.*, Bowler, R.**, Lebel, J., Hudnell, K.*** Université du Québec á Montréal, Canada, *McGill University, **San Francisco State University, ***United States Environmental Protection Agency, USA

Fish in the St?Lawrence Lakes (SLL) contain potential neurotoxins, including methylmercury, arsenic, lead, PCBs, DDT and DDE. Biological indicators of these substances among SLL fishermen have shown that concentrations do not surpass recommended levels. As part of a study on early neurotoxic effects of environmental exposures in Southwest Quebec, we surveyed SLL fish?eating during summer and winter. The study population was selected through the Québec National Health Plan, using a stratified, random design. After excluding persons with potential workplace exposure, we retained 304 women and men between the ages of 20 ? 69 years, who underwent a neurofunctional test battery, a neurologic examination and filled out a questionnaire containing socio?demographic information and symptoms. A total of 121 (40.3%) reported eating SLL fish, while 179 (59.7%) reported never eating SLL fish. There was no difference in age and smoking status between the two groups, although SSL fish?eaters consumed significantly more alcoholic beverages (p<0.01). Comparison of neuro?outomes (ANOVA) revealed that the SLL fish?eaters present significantly (p<0.05) greater motor slowing, poorer results on certain tests of memory and attention and higher scores on the Confusion Scale of the Profile of Mood States Test, taking into account age, education and alcohol intake. There appears to be a dose?effect: those who consume fish year round present poorer results than those who consume fish during one season, who have poorer results than those do not consume SLL fish. These findings suggest that nervous system alterations are associated with fish?eating from the SLL, and that current guidelines for fish consumption may be inadequate to prevent adverse effects. Since a large number of neurotoxins are absorbed with the fish, there may be an additive or synergistic effect. More studies are needed to determine which substance(s) account for these effects and long?term health consequences. Funded by USEPA Cooperative Agreement #CR822790, but does not necessarilyreflect the Agency's views

O-43 AN UNDERSTANDING OF MECHANISM IN NEUROTOXICOLOGYIS AN ACADEMIC LUXURY NOT NEEDED FOR PRACTICAL DECISION MAKING - FOR THE MOTION Ladefoged, O. Institute of Toxicology, National Food Agency, Soborg, Denmark

Commission Directive 93/67 EEC and Commission Regulation No. 1488/94 require that a human health risk assessment can be carried out on new substances or on priority existing substances, respectively. The risk assessment should proceed in the following sequence: Hazard identification, dose response assessment, exposure assessment, and risk characterisation. Within the field of neurotoxicology special problems arise in each of these steps, which may be different from problems in risk assessment of other endpoints than the nervous system. Very often the mechanism of the neurotoxic effect is not understood. Most neurotoxicological risk assessment is limited to qualitative hazard identification. In developmental neurotoxicity the qualitative and quantitative assessment of effects induced by neurotoxicity of ethanol, methylmercury, lead, PCB and drugs of abuse have been carefully studied. For several well©known neurotoxic compound such studies have not been performed. It is not always possible for the regulators to wait for mechanistic explanation but pragmatic solutions have to be found. For the hazard identification of new chemicals or drugs the risk assessment is mainly based on animal studies. For several well-known neurotoxicants the rat is a very poor model for the neurotoxic effects or symptoms seen in humans. The rat may be insensitive to the chemical, or it may not possess functions that are comparable to the affected function in humans. Dose response curves obtained in animal studies with neurotoxic endpoint are not relevant in human. How can this be used in a quantitative risk assessment? To obtain better data to be able to perform the risk assessment EU Technical Guidance Document on Human Health Risk Assessment of New and Existing Substances contain a testing strategy for repeated-dose studies. The most appropriate methods for further investigation of neurotoxicity should be determined on a case-by-case basis, guided by the effects seen in the standard systemic toxicity test and from SAR-based prediction. In my opinion this is a very crucial point in the future to select valid methods just to avoid performing animal studies where no effect in fact would be expected. On the other hand it is important for the competent authorities to react more pragmatic in toxicology if some neurotoxicity is seen in animal studies even if the mechanism is not known. An example of a risk characterisation of toluene is given. Conclusions about the neurotoxicity of toluene might be very difficult or impossible to reach if only animal data are used for the risk assessment. For another solvent, n-hexane an excellent risk assessment can be performed, based on animal data, if toxicokinetic differences between rat and human is taken into account. The two examples show that risk assessment of neurotoxic chemical, limited to the no-observed-effect level and uncertain factors cannot accurately encompass the diversity of neurologic responses to injury.

O-44 AN UNDERSTANDING OF MECHANISM IN NEUROTOXICOLOGY IS AN ACADEMIC LUXURY NOT NEEDED FOR PRACTICAL DECISION MAKING: AGAINST THE MOTION. Mattsson, J.L. The Dow Chemical Company, Health and Environmental Research Laboratories, Midland, Mitchigan, USA

Food for thought! Regulation of industrial chemicals has emphasized the most sensitive organ of the most sensitive species, then added layers of safety factors. Mechanisms have not been a key element in current risk assessment. This approach has been simple compared to the task we now face. Traditionally, large safety factors or banishment have been the rule. But, what about natural chemicals? As scientists, we know that there are no toxicological differences between natural and synthetic chemicals. Structure and dose are the driving features, and not 'who' made the chemical. Natural chemicals are very plentiful and often more potent than synthetic chemicals. A precarious balance exists between a plant's having sufficient levels to survive its hostile environment, and have low enough levels for us to eat them. Large safety factors and banishment are not options. Simply, we will have to be more knowledgeable about what we are doing. Sensible regulation requires an understanding of the mechanisms of toxicity, not just to determine why chemicals are toxic, but to explain why chemicals are safe.

O-45 THE ROLE OF OXIDATIVE STRESS ON CHOLINERGIC AND GLUTAMATERGIC INDUCED EXCITOTOXICITY Savolainen, K.M., Loikkanen, J., Naarala, J. Dept. of Pharmacology and Toxicology, University of Kuopio, Dept. of Environmental Medicine, National Public Health Institute, Kuopio, Finland

Excessive cholinergic and glutamatergic brain stimulation result in seizures, excitotoxicity and neuronal damage. It has been suggested that reactive oxygen species (ROS) production could be involved in both events. We have found that stimulation of human SH-SY5Y neuroblastoma cells with a cholinergic muscarinic agonist, carbachol, greatly increases the production of ROS without affecting intracellular GSH or cell viability. Cholinergic ROS production could be prevented with a protein kinase C (PKC) inhibitor and superoxide dismutase. Thus, PKC may play a role in cholinergic ROS production, and the ROS produced by carbachol may be superoxide anion. A salient feature of glutamate-induced excitotoxicity in neuroblastoma cells is also production of ROS, prevented with a specific PKC inhibitor and amplified with lead. Both findings suggest a role for PKC in glutamatergic ROS production in these cells. In murine hypothalamic GT1-7 cells, glutamate increased ROS production, but this production could not be prevented with a PKC inhibitor. Lead amplified glutamate-induced ROS production without extracellular calcium, and this amplification could be partially prevented with a PKC inhibitor, again suggesting a role for PKC. Glutamate also decreased intracellular GSH levels without affecting viability of GT1-7 cells. Lead amplified glutamate-induced GSH depletion and decreased cell viability together with glutamate; a PKC inhibitor did not prevent these effects. These results suggest a complex role for PKC in receptor-mediated ROS production and excitotoxicity in neuronal cells. Supported by the Academy of Finland.

O-46 NEUROTOXICITY OF AMMONIA AND OF GLUTAMATE: MOLECULAR MECHANISMS AND PREVENTION Felipo, V., Hermenegildo, C., Montoliu, C., Llansola, M., Minana, M.D. Instituto de Investigaciones Citológicas, Valencia, Spain

Ammonia is the main responsible for the pathogenesis of hepatic encephalopathy. We found that acute ammonia toxicity is mediated by activation of NMDA receptors. Chronic moderate hyperammonemia prevents acute ammonia toxicity in rats. Ammonia added to cultured neurons impairs NMDA receptor function (due to decreased protein kinase C-mediated phosphorylation) and prevents glutamate (Glu) neurotoxicity. Compounds that prevent ammonia toxicity in mice (e.g. carnitine) also prevent Glu toxicity in cultured neurons. These compounds did not prevent activation of NMDA receptor nor the rise of Ca2+. They interfere with subsequent steps in the toxic process. The protective effect of carnitine is mediated by activation of metabotropic glutamate receptors. Agonists of mGluRs, specially of mGluR5, prevent Glu toxicity. Agonists of muscarinic receptors also prevent Glu toxicity and it seems to be an interplay between muscarinic and metabotropic Glu receptors in the protective effect. We try to identify intracellular events involved in the process of neuronal death. It is known that the rise of Ca2+ is an essential step. Glu leads to depletion of ATP; some compounds (e.g. carnitine) prevent Glu-induced neuronal death without preventing ATP depletion: additional events are required for neuronal death. Glu induces activation of Na+/K+-ATPase, which could be involved in the toxic process. Inhibitors of protein kinase C, calcineurin or nitric oxide synthase prevent Glu toxicity. Our results indicate that Glu toxicity can be prevented at different steps or by activating receptors coupled to transduction pathways interfering with the toxic process. Agents acting on these steps could prevent excitotoxicity in vivo in animals.

O-47 MECHANISMS OF PRIONSc- AND HIV-1 gp120 INDUCED NEURONAL CELL DEATH Schröder, H.C., Perovic, S., Ushijima, H.*, Müller, W.E.G. Institut für Physiologische Chemie, Johannes Gutenberg-Universität, Mainz, Germany, *Institute of Public Health, Tokyo, Japan

The scrapie prion protein, PrPSc, which is the abnormal isoform of the cellular prion protein, PrPC, is the causative agent of a number of transmissible neurodegenerative diseases in humans and animals. In vitro experiments revealed that PrPSc induces apoptosis of rat cortical neurons. This effect was also observed in experiments when the PrP fragment PrP106-126 was added to the neurons. Significant toxicity (70% of cell viability) was measured already at a concentration of 50 µM of the peptide, while at higher concentrations this effect increases to 70% during a 9 days of incubation. The toxic effect displayed by PrP could be blocked by NMDA receptor antagonists. Concomitantly with the reduction of cell viability PrP106-126 caused a significant drop of intracellular glutathione level in neuronal cells. The level of the anti-apoptotically acting proto-oncogene product Bcl-2 increased in samples, treated with 100 µM PrP106-126 after 3 and 6 days of incubation; after 9 days the Bcl-2 level dropped to 82%. Rat cortical cells also undergo apoptosis after incubation with the HIV-1 coat protein gp120 in vitro. This effect was abolished by MK-801 and other NMDA antagonists. We found that in response to exposure to gp120 cells react with an increased release of arachidonic acid (AA) via activation of phospholipase A2. This process was not inhibited by NMDA receptor antagonists. To investigate the role of AA on the sensitivity of the NMDA receptor towards its antagonist, low concentrations of NMDA were co-administered with AA. This condition enhanced the NMDA-mediated cytotoxicity. Administration of mepacrine reduced cytotoxicity caused by gp120. We conclude that gp120 causes an activation of phospholipase A2, resulting in the increased release of AA, which may in turn sensitize the NMDA receptor.

O-48 NITRIC OXIDE SYNTHASE AS A MOLECULAR TARGET FOR NEUROTOXICANTS Desaiah, D. Department of Neurology, The University of Mississippi Medical Center, Jackson, MS, USA

Nitric oxide synthase ( NOS ) catalyzes the endogenous synthesis of nitric oxide in many cell types of several organs of different species. In mammalian systems three different isoforms of NOS have been isolated,purifird and well characterized. The two isoforms i.e. n-NOS and e-NOS are calcium requiring enzymes constitutively expressed and regulate a variety of physiological functions in brain. The physiological NO acts as a messenger molecule for the actions of hormones and neurotransmitters including the glutamate neurotransmission thereby regulating the hippocampal long - term potentiation. A number of environmental neurotoxicants such as polychlorinated biphenyls, chlordecone, lead, and trimethyltin have been shown to modulate neurobehaviour of animals and impairment of cognitive function in children. Our laboratory data show that these neurotoxicants are potent modulators of brain NOS suggesting that NOS may be a molecular target for these neurotoxicants. An attempt will be made in this presentation to construct a relationship between the inhibion of brain NOS and impairment of cognitive function by the neurotoxicants.

O-49 BIOCHEMICAL MECHANISMS UNDERLYING TOXICITY OF EXCITATORY AMINO ACID L-ß-OXALYLAMINOALANINE PRESENT IN LATHYRUS SATIVUS Ravindranath, V., Sriram, K. Department of Neurochemistry, NIMHANS, Bangalore, India

Ingestion of chick pea (Kesari dal) from the plant Lathyrus sativus leads to a progressive clinical disorder involving the cortico-spinal motor tracts, commonly called Lathyrism. The toxic principle in the plant has been identified as L-ß-oxalylaminoalanine (L-BOAA). The toxicity of L-BOAA has been linked to its action on glutamate receptor, since L-BOAA is an agonist of AMPA receptor, a subclass of glutamate receptor. Exposure of sagittal slices of mouse brain to low concentrations of L-BOAA results in discernible damage to neurons and loss of mitochondrial Complex I activity, which is abolished by prior exposure to glutamate antagonists. Other glutamate agonists, such as NMDA, AMPA, quisqualate etc. also inhibit Complex I although at a much higher concentration. Administration of a single dose of L-BOAA to mice results in inhibition of Complex I in select regions of the CNS (lumbo-sacral cord, motor cortex etc.) while other regions (thoracic cord, occipital cortex etc.) are unaffected. Chronic administration of L-BOAA to mice for 45 days also resulted in the selective inhibition of Complex I in the lumbo-sacral cord and motor cortex while the thoracic cord was unaffected. The inhibition of Complex I caused by exposure to L-BOAA both in vitro and in vivo could be attenuated/abolished by disulfide reducing reagents such as glutathione, glutathione ester, dithiothreitol and a-lipoic acid. The involvement of thiol oxidation in the inhibition of Complex I was also demonstrated by the fact that L-BOAA administration caused a sustained loss of glutathione in the lumbosacral cord. The biochemical mechanism underlying neurotoxicity of L-BOAA involves thiol oxidation and inhibition of mitochondrial respiration as downstream events following receptor activation.

O-50 PCB LEVELS IN BRAIN TISSUE FROM NINE STILLBORNS OF VARYING GESTATIONAL AGE IN RELATION TO LEVELS IN LIVER AND ADIPOSE TISSUE Lanting, Caren I., Boersma, E.R. Dept. of Obstetrics and Gynaecology, Perinatal Nutrition and Development Unit, University of Groningen, Groningen, The Netherlands

Higher levels of prenatal exposure to polychlorinated biphenyls (PCBs) have been found to adversely affect brain function in infancy and childhood. We collected brain-, liver-, and subcutaneous adipose tissue of nine stillborns whose gestational age varied from 17 to 40 weeks. Depending on the total brain size, tissue was sampled from the parieto-temporal or parietal area. The tissue samples were analyzed for PCBs. Ratios of PCB levels in brain to PCB levels in liver- and adipose tissue were calculated. The PCB levels and the ratios in brain-, liver-, and adipose tissue were sorted by the gestational age of the fetus, and visually inspected for trends. In four fetuses the presumed cause of death was placental insufficiency. Other causes were abortus provocatus, ventricular bleeding, and intrauterine pneumonia. In two cases the cause of death remained unknown. On a fat weight basis, the ratio of the PCB level in the brain to the PCB level in subcutaneous fat tissue ranged from 0.10 to 0.55. For the liver, this ratio varied between 0.48 and 1.03. Levels in adipose tissue are in the same range as levels that have previously been found in breast milk obtained from Dutch mothers in their second week after delivery. Although definite conclusions can not be drawn from data concerning such a small group of fetuses, the PCB levels in brain-, liver-, and fat tissue do not seem to vary with gestational age.

O-51 NEUROFILAMENT METABOLISM AND 2,5-HEXANEDIONE Heijink, Elisabeth, Scholten, S.W., Wolterman, R.W.*, Bolhuis, P.A.*, de Wolff, F.A. Coronel Institute, Dept. of Human Toxicology, *Dept. of Experimental Neurology, Academic Medical Centre, University of Amsterdam, The Netherlands

Exposure to 2,5-hexanedione (2,5-HD), the neurotoxic metabolite of n-hexane, induces accumulations of neurofilaments (NF) within the neuroblastoma cell line SK-N-SH. Further, high molecular weight cross-linking products of the NF-proteins (HMW-NF) have been demonstrated after exposure to 2,5-HD. We questioned whether 2,5-HD-induced NF-accumulation and cross-linking affects NF-metabolism. We isolated NF-proteins from cells treated with 10 mM 2,5-HD for 3 days, and did in vitro degradation experiments using calpain. All three NF-proteins were degraded as fast as NF-proteins from control cells. In addition, the same was true for the HMW-NF. Metabolic labelling studies were set up to examine intracellular NF-metabolism in the presence of 5 mM 2,5-HD. Cells were pulsed with [35S]-methionine or [33P]-orthophosphate for 3 hrs, and chased for up to 72 hrs in the presence 5 mM 2,5-HD; or cells were first incubated with 2,5-HD and then pulse-labelled. SDS-PAGE and autoradiography of immunoprecipitated NF-proteins revealed that NF-turnover and (de)phosphorylation proceeded identical in exposed and control cells. HMW-NF were present in immunoprecipitates of [33P]-labelled cells exposed to 2,5-HD for 48-72 hrs, indicating that phosphorylated NF were cross-linked, and HMW-NF were still phosphorylated. In contrast, HMW-NF were not detected within 48 hrs after [35S]-labelling of the cells, suggesting that 2,5-HD does not cross-link newly synthesized NF. Taken together, these experiments revealed that 2,5-HD induces accumulation and cross-linking of NF-proteins without disturbing NF-metabolism.