INA 6 - Posters

P-1 A STUDY OF EFFECT OF LEAD ON EVENT-RELATED POTENTIALS AMONG LEAD-EXPOSED WORKERS Hirata, M., Kosaka, H., Yoshida, T. Dept. of Occupational Health, Osaka Prefectural Institute of Public Health, Osaka, Japan

In order to clarify the effect of lead on cerebral higher function , lead-exposed workers (Pb group) and control workers (C group) were examined for event-related potentials. Fourteen lead-exposed workers with mean age 57.0 years (SD=4.26, range 48-64; concentration of whole blood ranged from 33 to 106 mg/dl with mean 58.6 and SD 28.6 mg/dl) and age-matched control workers with mean age 57.0 years (SD=4.26, range 48-65) were examined. Visual P300 was recorded by button pushing to target image (minute checkerboard pattern, 20%), and NO-GO potential by no button pushing to target image (same above, 50%). Latencies of P300 in Pb group (475±46.0 msec) were significantly delayed compared with those in C group (407±42.4 msec, p<0.01 by Student's t test). Latencies of NO-GO potential in Pb group (150±9.24 msec) tended to delay compared with those in C group (144±15.9 msec), but not significant. The findings suggest that lead exposure involves cognitive and attention function of the cerebral higher functions, but not the suppression function of movement of those.

P-2 NO EVIDENCE OF LEAD POISONING IN SCROLL SCRIBES Finkelstein, Y., Amitai, Y.*, Erlichmann, M.**, Zhao Wei***, Hod, I.**, Rosen, J.F.*** Shaare Zedek Med. Ctr., *Hadassa Mount Scopus Med. Ctr., **The Hebrew University of Jerusalem, Israel, ***Division of Environmental Sciences, Albert Einstein College of Medicine, New York, USA

Scrolls of the Bible and Jewish religious articles (Torah, Tefilim, Mezuzo) are traditionally scribed on parchment with a special ink prepared in time-honoured methods. Historically it contained lead sulphate in addition to other ingredients, and prepared by heating the mixture to transform it into a dark ink. Although esoteric, this profession is deeply rooted and even popular in the recluse Jewish extremely orthodox communities in Jerusalem, where a few hundreds scribes live. The possibility of occupational exposure to consequential lead poisoning has been clinically evidenced during the last decade, in twelve cases. In all those cases, the home-made ink has been proved to contain lead. The purpose of this study was to determine whether these sporadic reports reflected a general occupational problem or were merely episodic. Forty six scribes were clinically examined, after filling a demographic-occupational health data questionnaire. Blood samples were drawn and lead blood levels were determined using a graphite furnace atomic absorption spectrometry. All the examined scribes were males (age 29-59 years) and have practiced their craftsmanship for 1-20 years. They have worked 15-40 hours a week (median - 26 hours); all of them were non-smokers. Their medical history included complaint that could be related to lead poisoning and their physical examinations were within normal limits. In all 46 cases, blood lead levels were far below the toxic threshold, ranging between 0-6 mg%. Their hemoglobin ranged between 12.1-16.3 g%. We conclude that the contemporary practice of the traditional art of scroll scribing does not expose its practitioners to the hazard of lead poisoning

P-3 NEUROBEHAVIORAL EFFECTS DURING AND AFTER LOW DOSE EXPOSURE TO TETRAALKYLLEAD Seeber, A., Kiesswetter, E., Blaszkewicz, M. Institute of Occupational Physiolgy at the University of Dortmund, Germany

Tetraalkyllead compounds are produced as antiknock additives for gasoline. Exposed workers showed with increasing tetraalkyllead concentrations (above 50 µg/m3 Pb in the air or 30 µg/l diethyllead (Et2Pb2+) in urine) an enhanced level of neurasthenic symptoms and tremor. In a different study with lower exposure (10 µg/l Et2Pb2+ in urine) significant correlations between total lead in urine and neurological symptoms and between Me3Pb+ (trimethyllead) in urine and coding performance (digit symbol test) were shown. Generally, a higher neurotoxicity of organolead compared with inorganic lead is assumed. Two further examinations were carried out in that plant where the last mentioned results were obtained in the first step (t1 after 14 ± 6 years of exposure, n=38): the t2 study was performed 13 months and the t3 study 19 months later (n=24). In t3 two groups of workers were investigated, one who disassembled the factory after cessation of the production (n=10) and another who had left the factory after t2 and had no further exposure (n=14). Both, total lead in urine and diethyllead in urine decreased significantly from t1/t2 to t3 in the group who had left the firm, whereas they increased in the disassembly group. These changes have no correspondence within the neurobehavioral performance data. However, there are corresponding changes of the neurological symptoms measured by questionnaire. The enhanced level of symptoms in the disassembly group is accompanied with the opinion that the state of health did not change essentially. The decrease of the symptoms in the leaving group goes along with the estimation that the state of health had improved. The follow up study shows that the dose-response relationship of t1 could not be proved repeatedly. Nervertheless, concerning neurotoxic symptoms, differences between the groups could be observed under low level exposure.

P-4 NEUROBEHAVIORAL PERFORMANCE IN YOUNG ADULTS TWENTY YEARS FOLLOWING CHILDHOOD LEAD EXPOSURE: THE BUNKER HILL EXPERIENCE Stokes, L.D., Letz, R.*, Gerr, F.*, Kolczak, M., Mcneill, F.E.**, Chettle, D.R.**, Kaye, W.E. CDC/ATSDR, Atlanta, *Emory University, Atlanta, **McMaster University, Ontario, Canada

A cohort population was identified of 917 young adults 19-29 years of age who resided, during childhood, near the Bunker Hill Smelter in Silver Valley, Idaho, USA. Smelter emissions had exposed the surrounding community to elevated lead levels when subjects were 9 months to 9 years of age. The purpose of the investigation was to determine whether neurobehavioral effects differed between the lead exposed group and referents. A cross-sectional sample of the cohort (N=281) and referent group (N=287) frequency matched on age was studied. The referent group resided 60 miles away without residential proximity to smelter emissions. Exposure assessment of lead body burden was determined by K X-ray fluorescence of the tibia. The exposed group had higher (p<.001) bone lead concentration than the referents (difference 4.0±0.7 [S.E.M.] mg Pb/g bone mineral). Tests from the Neurobehavioral Evaluation System (NES) - finger tapping, hand-eye coordination, symbol-digit substitution, vocabulary, serial digit learning, and mood - were administered. In addition postural sway, vibration sensitivity, manual and electrophysiologic tests were performed. Neuropsychiatric symptoms from the Q16 (The Scandinavian Questionnaire) were also collected. Lead exposed young adults performed poorer than referents, excluding electrophysiologic tests. Deficits in cognitive performance showed a dose-response relationship with increasing bone lead concentration. Neuropsychiatric symptoms were significantly more prevalent in the exposed when compared to referents. Results show that neurobehavioral effects exist twenty years following lead exposure during childhood.

P-5 EVALUATION OF IQ- AND NON-IQ-MEASURES IN ASSOCIATION WITH LEAD AND MERCURY IN SIX YEAR OLD CHILDREN: CONTROL FOR VISUAL FUNCTIONS Winneke, G., Altmann, L.*, Hennekes, U., Krämer, U.**, Sveinsson, K.*, Turfeld, M.***, Walkowiak, J., Weishoff-Houben, M.****, Zovkic, U. Divisions of Biological Psychology, *Neurotoxicology, **Epidemiology, ***Analytical Chemistry of the Medical Institute of Environmental Hygiene at the Heinrich-Heine-University of Düsseldorf, ****Institute of Hygiene & Environmental Medicine of RWTH Aachen, Germany

Aim: To compare the differential vulnerability of psychometric intelligence and sensorimotor functions in relation to background levels of Pb and Hg after adjustment for relevant confounders or covariates, including visual functions. Methods: Lead-concentrations in blood (PbB) and urinary Hg-excretion (HgU) were measured in 384 six year old children. Two WISC-subtests, Vocabulary = V and Block Design = BD, as well as five subtests from NES2, namely Pattern Comparison/Memory, Finger Tapping, Simple Reaction Time, and CPT (child version; Dahl et al., 1996), were measured as target variables. Visual acuity (TITMUS) and contrast sensitivity (FACT) were measured, as well. Results: The median PbB was 42.5 µg/l (range: 14-174 µg/l) and the median HgU was 157 ng/24 h (20-2830 ng/24 h). No significant associations were established between HgU and any of the neurobehavioral target variables. Significant negative associations were found for PbB, however, namely with CPT miss- (NR) and false alarm-rates (FP; p £ 0.05), and borderline ones (p < 0.10) for WISC-V and for the combined WISC-measure, but not for WISC-BD. Contrast sensitivity was a strong covariate for most NES-tests, namely for Pattern Memory, Finger Tapping, Simple RT and CPT-variables NR and FP.

P-6 MYOPATHY: A POSSIBLE EFFECT OF CHRONIC LOW LEVEL LEAD EXPOSURE Buchheim, Katherina, Soltenburg-Didinger, G., Lilienthal, H.*, Winneke, G.*,Wiegand, G.* Inst. of Neuropathology, Clinic Benjamin Franklin, Free University Berlin, *Medical Institute of Environmental Hygiene, Heinrich-Heine-Univ., Düsseldorf, Germany

Morphological changes in the central nervous system and other organs have been reported in numerous studies investigating low level lead exposure. To date, however, there are no investigations on the effect of low level lead exposure on striate muscles, although varying neuromuscular changes in different species after subchronic exposure are known for years. Rhesus monkeys were exposed pre- and postnatally to lead acetate in diet (0 or 350 ppm or 600 ppm) over 9 years, followed by a lead free period of 32 months. During this time blood lead levels of treated animals dropped to control levels. No signs of muscular dysfunction were evident. To elucidate neuromuscular pathomorphology frozen sections of the vastus medialis were processed for routine and enzymohistological stainings (HE, Sudan, Gömöri, NADH, ATPase). Resin histology was processed for electron microscopy. Morphometric analysis was made with commercial software (CAMoM,1994). Light microscopy revealed dose-related signs of myopathy in the lead-exposed groups. The scatter of fibre diameters was increased, splitting fibers and internal nuclei were more frequent. Fibers became separated from each other by copious endomysial connective tissue. Ultrastructural examination showed hydropic mitochondria and massively dilated sarcotubular system in myocytes in the 600 ppm group. Dose-related ubiquos extracellular collagen deposition increased. A heavy fibrosis was seen in the 600 ppm group. These findings are interpreted as myopathic reaction due to chronic low level lead exposure, while there were no signs of neurogenic lesion. It remains unknown how the fibrosis developed. A primary fibrosis could be based upon a developmental delay of satellite cells (expressing metalloproteases for collagen-metabolism). Lead is known to inhibit regular development in many ways if exposition has started prenatally. As the skeletal muscle is a common target of toxicity, the myotoxic effects of chronic low level lead exposure comes into question.

P-7 GFAP GENE EXPRESSION IS ALTERED IN YOUNG RATS FOLLOWING DEVELOPMENTAL LOW LEVEL LEAD EXPOSURE Partl, S., Schaeper, F.**, Herbst, H.***, Hummel, M.*, Stoltenburg-Didinger, G. Institutes of Neuropathology and *Pathology, University Hospital Benjamin Franklin, Free University Berlin, Institutes of Pathology, **University Hospital Rudolf Virchow, Humboldt Univ. Berlin, ***Univ. Hospital Eppendorf, Hamburg, Germany

The effects of lead were investigated in 17 to 21 days old Wistar rats exposed to lead continuously during gestation and postnatal development. The control animals were the offspring of and suckled by unexposed mothers. Radioactive in situ hybridization was performed with a riboprobe that specifically detects GFAP RNA. Measurement of the gray values in the autoradiographies revealed significantly reduced signals for GFAP RNA in the gray matter of hippocampal formation of lead exposed rats compared to the unexposed controls. The contralateral brain halves of the same animals used in this study were previously involved in a LTP measurement study, which revealed LTP impairment in 15-21 days old rats. The coincidence of LTP impairment and GFAP RNA reduction following low level lead exposure may be indicative for a glial-neuronal crosstalk in which structural differentiation of the glia is regulated by neuronal information processing. NMDA receptor subunits were recently identified on the surface of astrocytes. This finding indicates that glia cells are able to discern synaptic activity. Lead inhibits the neuronal NMDA receptor resulting in LTP impairment. This LTP impairment in the synaptic space may result in a lower excitation of the glial NMDA receptors. This "electrophysiological silence" affects the GFAP RNA household in the astroglia, which is possibly coupled with and regulated by NMDA receptor activity of the astrocytes.

P-8 LEAD AS A RISK FACTOR? ALTERATIONS IN GLUTAMATERGIC AND DOPAMINERGIC FUNCTION IN MESOCORTICOLIMBIC SYSTEMS AND THEIR RELATIONSHIP TO BEHAVIORAL TOXICITY Cory-Slechta, D.A., Richfield, E.K.*, McCoy, L.M.**, Zuch, C.L., O'Mara, D. Departments of Neurobiology and Anatomy, *Neurology, and **Psychiatry, University of Rochester Medical School, Rochester, NY, USA

Chronic low-level postweaning Pb exposure in rats produces a pattern of neurochemical changes comprised of hypoglutamatergic function in conjunction with hyperdopaminergic function that is observed in mesolimbic but not nigrostriatal systems. Autoradiographic studies revealed that Pb decreases dopamine (DA) binding sites in nucleus accumbens (NAC), consistent with excess synaptic DA in this region, but has no effect in dorsal striatum (DS), even over 12 months of exposure. Consistent with these findings, in vivo electrochemistry studies show Pb-induced increases in KCl-evoked DA overflow in NAC but not DS. Pb decreases binding of the non-competitive NMDA receptor antagonist across brain regions. Since MK-801 binding is considered a marker of NMDA activity, these data are consistent with widespread hypoglutamatergic function. Behavioral studies indicate that these changes in DA and glutamatergic (GLU) function in NAC underlie the changes in fixed interval (FI) schedule-controlled behavior produced by Pb in experimental animals. Irreversible blockade of DA receptors in NAC but not DS results in marked alterations in FI performance in normal rats, indicating the critical role of NAC in mediating FI performance, and microinjection of MK-801 into NAC produces changes in FI schedule-controlled behavior with marked similarities to those produced by Pb. It is notable that a remarkably similar pattern of neurochemical changes is currently believed to underlie schizophrenia. Changes in both DA/GLU mesocorticolimbic function are also thought to mediate behavioral sensitization to the motor activity effects of psychostimulants, a phenomenon believed to be involved in drug abuse and addiction and in psychosis. These findings suggest that Pb may be a risk factor for schizophrenia or for drug abuse and addiction, or for other behavioral and or neurological disturbances associated with mesocorticolimbic systems. ES05017, ES05903.

P-9 LEAD NEUROTOXICITY: THE ROLE OF GLUTAMATE, EXTRACELLULAR CALCIUM AND REACTIVE OXIGEN SPECIES Loikkanen, J.J., Naarala, J.*, Savolainen, K.M.* National Public Health Institute, Laboratory of Toxicology, *Department of Pharmacology and Toxicology, University of Kuopio, Kuopio, Finland

Effects of lead acetate (1 mM PbAc), L?glutamate (1 mM Glu) and their combination on the production of reactive oxygen species (ROS), the levels of intracellular glutathione (GSH) and cell viability were studied in mouse hypothalamic GT1?7 cells in the presence and absence of 1.3 mM extracellular calcium. PbAc alone did not affected ROS production, but depleted GSH and decreased cell viability. Glu alone increased ROS production and depleted GSH, but had no effect on cell viability. The presence or absence of extracellular calcium did not modify the effects caused by PbAc and Glu alone. PbAc amplified strongly Glu?induced ROS production, but only in the absence of extracellular calcium. Glu and PbAc together depleted levels of GSH and decreased cell viability more markedly than these compounds alone, both in the presence and absence of extracellular calcium. Neither protein kinase C (PKC) inhibitor (1 µM Ro 31?8220) nor superoxide dismutase (100 µg/ml SOD) modified effects of Glu and PbAc on GSH depletion and cell viability, although PKC inhibitor and SOD partially blocked amplification of Glu?induced ROS production by PbAc in the absence of extracellular calcium. These data suggest that an excitatory neurotransmitter, glutamate, increases lead neurotoxicity, preferentially through mechanisms other than ROS production. Supported by the Academy of Finland.

P-10 LEAD IONS CHANGE THE CONDUCTION VELOCITY OF ACTION POTENTIALS NOT ONLY BY AN INTERACTION WITH CA2+ -CHANNELS Zartl, M., Hoyer, J. Institute of Neurophysiology, University of Vienna, Wien, Austria

Ca2+- channels are said to be major target sites for the action of heavy metal ions. Many heavy metals, in particular Pb2+, interfere with conduction velocity (CV) of action potentials (APs) in peripheral nervs. We investigated the influence of Pb2+ on the CV by simultaneous intracellular recordings of the soma and the axon from giant cells of Aplysia californica. Registration of APs in the phase plane mode (dV/dt versus V) gives insight into activation of inward current and Ca2+ - dependent K+- current. The latter current does not exist in axonal APs. Pb2+ (2µM) was added to artificial sea water, used as perfusate for isolated ganglia. In addition experiments were performed with variable Ca2+ concentration, alternatively Co2+ was added to block Ca2+- currents. Pb2+ enhances the activation of inward current, and thus reduces AP-duration. In this respect, the action of Pb2+ is mimiced by Co2+ as well as by low concentration of Ca2+ in the perfusate. The effect of Pb2+ is, as expected antagonized by high Ca2+ concentrations. So far the results show opposite effects of high Ca2+ concentrations on the one hand and the action of either low Ca2+, the Ca2+ - blocker Co2+ or Pb2+ on the other hand. CV of APs was calcu-lated from the latency between the AP registered from the soma and the one recorded simul-taneously from the axon and the distance measured between the two recording electrodes. CV was enhanced by Pb2+, whereas it was reduced by Co2+. Since Ca2+- channels do not contribute to the propagation of APs in the peripheral axon and the Ca2+ - blocker Co2+ has the opposite effect to Pb2+, we conclude that Ca2+- channels cannot be the primary target site for the action of Pb2+ on the CV of Aps. Supported by Österr. Nationalbank/Jubiläumsfonds Proj. 6372

P-11 GENETIC EFFECTS IN NEOCORTICAL CELLS OF RATS TRANSPLACENTALLY EXPOSED TO MERCURY COMPOUNDS De Visscher, G., Szelecki, T.*, Szente, M.*, Kirsch-Volders, M. Laboratory for Human Genetics, Vrije Universiteit Brussel, Brussels, Belgium, *Department of Comparative Physiology, Attila József University, Szeged, Hungary

The results presented here are a preliminary genetic study, part of a broader EC-COPERNICUS research project on the neurotoxic effects of methyl-mercury in the neocortex of the rat: cellular electrophysiological and mutagenicity studies. Adult Wistar rats were exposed to HgCl2 in drinking water (0, 0.8 and 1.6 mg/kg/day) from week 1 to week 6 or 7. Four female and 1 male rats were placed together on week 1 and left together until delivery. Three cages were made available per experiment (one control and two treated). The young rats from two females per cage were studied at day l, 2, 3, 4 ,12 and/or 13 after birth. The pups were killed by Nembutal overdose and the nuclei from the neocortex were isolated by a modification of the method described in Singh et al. (1995). Purity of the fraction was controlled under microscope. The isolated nuclei were further analysed with the Comet assay (Belpaeme et al.,1996; Van Goethem,1997) which allows a cell by cell quantification of DNA breakage. The results measured as tail length and tail moment indicate that a transplacental chronic exposure of pregnant female rats to HgCl2 did not induce a significant DNA damage in neocortical cells of young rats. An age-dependent variation of tail moment and of highly damaged (apoptotic) cells was observed. Similar studies with CH3HgCl are in progress. Singh et al.1995. Mutation Res 345,191-196 Belpaeme et al.1996. Mutagenesis 1 l, 4, 383 - 389 Belpaeme et al.1996. Mutagenesis 11, 5, 485 - 492 Van Goethem et al.1997. Mutation Research, in press.

P-12 ELECTROPHYSIOLOGICAL EXAMINATION OF CHRONIC DEVELOPMENTAL EXPOSURE TO MERCURIC CHLORIDE IN RATS Szász, A., Szelecki, Tünde, Szente, Magdolna József Attila University, Dept. of Comp. Phys., Szeged, Hungary

Mercury compounds are known to cause neurologic impairment of developing central nervous system in humans and in several animal models. However, no detailed information is currently available concerning the changes in electrophysiological activity and epileptogenicity of the neocortex as a result of chronic, developmental exposure to mercury neither in human populations nor in animal models. In the present study female rats were exposed to HgCl2 in the drinking water. Exposure was begun a week before of mating and continued through gestation and the sucking period to the time of experiment. No gross evidence of maternal mercury intoxication was observed at any time during the study. Mercury exposed newborns weighed less than controls and the treated litters showed an increase in the pup death rate. Electrophysiological parameters of aminopyridine-induced cortical epileptic activity were measured in treated offsprings and compared to those of control animals of the same age. Epileptogenicity was significantly increased in treated offsprings, characterized by a shorter latency of seizure onset, increased duration of ictal activity, changes in the amplitude and frequency pattern of epileptic discharges with a strong tendency to generalization of epileptic activity. In addition in non-treated adult rats, ongoing aminopyridine-induced epileptic activity was highly facilitated after acute i.p. injection of HgCl2 solution. Many sites within the central nervous system can be potentially influenced by both direct and indirect effects of mercurials, contributing to increased epileptogenicity. More detailed in vivo studies are required to further elucidate the mechanisms responsible the changes in electrophysiological properties of the central nervous system.

P-13 INDUCTION OF APOPTOSIS BY MERCURY COMPOUNDS Monnet-Tschudi, F., Eskes, C., Honneger, P. Institute of Physiology, University of Lausanne, Lausanne, Switzerland

Using aggregating brain cell cultures of fetal rat telencephalon as a model, we have shown that very low concentrations of mercury compounds applied chronically induced an early microglial reaction (Monnet-Tschudi et al.,Brain Res. 741, 52, 1996). We hypothetized that an induction of apoptosis by mercury compounds could be an indirect activating signal of the microglial reaction. Therefore aggregate cultures were treated during 10 days with mercury chloride and monomethylmercury chloride at non cytotoxic concentrations at two developmental periods: from day 5 to 15, corresponding to an immature stage and from day 25 to 35, corresponding to a mature stage. Apoptosis was evaluated by the TUNEL technique. In immature cultures, monomethylmercury chloride (10-10 M - 10-6 M) and mercury chloride (10-9 M - 10-7 M) caused a significant increase in the number of apoptotic cells. Double staining of apoptotic cells, and of microglial cells using the specific lectin Griffonia simplicifolia, showed no association. In mature cultures, mercury compounds did not affect the number of apoptotic cells. These results suggest that mercury compounds can induce apoptosis, but only in immature cultures exhibiting spontaneous apoptosis and that mercury-induced apoptosis is not an activating signal for microglial cells.

P-14 DIRECT EFFECTS OF MONOMETHYLMERCURY CHLORIDE AND OF TRIMETHYLTIN ON ISOLATED MICROGLIAL CELLS Eskes, C., Honneger, P., Monnet-Tschudi, F. Institute of Physiology, University of Lausanne, Lausanne, Switzerland

The earliest sign of neurotoxicity detected after low concentrations of mercury compounds and of trimethyltin was an increase in number and a clustering of microglial cells both in vivo and in vitro. In order to test whether the microglial reaction was due to a direct effect of the metal compounds or to an indirect effect via stress signals released by the neurons, we prepared cultures containing purified microglial cells. These cultures were treated during 10 days with monomethylmercury chloride (10-10 M - 10-6 M) and trimethyltin (10-10 M - 10-6 M). LPS (100 ng/ml) was used in control cultures to check the responsiveness of microglial cells. On isolated microglial cells, methylmercury chloride induced a change in the shape (appearance of rounded cells), as well as an increase in the intensity of the staining for the specific lectin Griffonia simplicifolia. At 10-6 M of methylmercury, a clustering of microglial cells was observed. Trimethyltin, in contrast, did not affect directly the microglial cells. Furthermore no proliferation of microglial cells was seen by autoradiography of 3H-thymidine, incorporated at the end of the treatment period with both metal compounds. The present results suggest that monomethylmercury, but not trimethyltin, may directly activate microglial cells.

P-15 NEUROTOXIC EFFECTS OF ALUMINIUM FROM DRINKING WATER Voroniuc, O., Mancas, G.*, Gavát, V., Palamaru, L.* University of Medicine and Pharmacy, *Medical Centre of Health Service and Management, Jassy, Romania

Neurochemical and morphological responses to chronic, oral aluminium admnistration have been studied in rats. Aluminium (0.3%) was added to drinking water of adult rats for four and eight weeks. We have searched the involvement of nonenzymatic hydroxylation processes, possibly mediated by free radical interactions. These studies show that chronic oral aluminium administration to rats has significant neurochemical and morphological consequences.

P-16 BEHAVIOURAL TERATOGENIC EFFECT OF ALUMINIUM EXPOSURE IN RAT OFFSPRING Gonda, Zsuzsanna, Lehotzky, Kornélia* Department of Toxicology, National Institute of Occupational Health, *Dept. of Comparative Physiology, Faculty of Science, Loránd Eötvös University, Budapest, Hungary

Aluminium has been proved to be a neurotoxic agent in a number of laboratory and epidemiological studies. In spite of this, there is very little information about the effect of prenatal Al exposure on cognitive functions. We investigated the behavioural teratogenic effects of 0, 2.5, 5 and 10 mg/kg daily s.c. Al-lactate exposure. Pups were tested for motor coordination, stress tolerance in a swimming test, behavioural patterns in an open field. Learning ability of the offspring was examined in different learning tasks. In the open field test, the exploratory activity diminished at the top dose of Al treatment. The motor coordination and the stress tolerance were not altered by prenatal Al exposure. Decreased learning ability of all treated pups was characterized by diminished performance and lengthened latency in an active avoidance responding task. In a social learning situation (observational conditioning) the performance of the active avoidance response improved, but the response latency was not shortened by this kind of facilitation. Long-term memory of pups exposed with the top dose of Al-lactate was impaired in one-trial passive avoidance task. In the 8 arm radial maze the spatial learning and the spatial memory of the offspring; treated with the high Al dose proved to be normal. The results confirm that behavioural teratogenic effects can be induced in rat pups treated with Al- lactate in utero.

P-17 COMPARATIVE MORPHOLOGICAL AND PHYSIOLOGICAL ASPECTS OF ALUMINIUM ACTIONS ON CENTRAL NEURONS AND NEURONAL SYNAPSES OF INVERTEBRATE AND VERTEBRATE ANIMALS Zsiros, V., Rojik, I., Kovács, T., Csóti, T., Erdélyi, L. Attila József University of Sciences, Department of Comparative Physiology, Szeged, Hungary

Aluminum is known as a biochemically toxic element ( Boegman and Bates, 1984; Forrester and Yokel, 1985) which can induce neurofibrillary degenerations in various neurons and encephalopathy (Crapper and Tomko, 1975; De Boni et. al., 1976; Yates et al., 1976). We studied the actions of aluminum compounds ( Al(OH)3, Al(OH)4-, Al(OH)2- , Al3+ ) on neurons and neuronal synapses of snail, Helix pomatia L. by use of morphological and electrophysiologycal techniques. Intracellular recordings of neuronal responses and synaptic events were analysed and the morphological alterations were followed. Responses of identified snail neurons were studied under current- and voltage-clamp conditions. Aluminum (100-900 uM at pH=6.9-4) first increased later decreased the amplitude of stimulus evoked postsynaptic responses. Both dose- and time-dependent actions of aluminum were analysed on resting membrane potential, input resistance and action potential parameters. Aluminum treatment significantly modified all the above mentioned parameters by causing modulation of the neuronal excitability.Furthermore, leak current and active membrane ionic currents were also increased or decreased in aluminum containing solutions. Aluminum treatment induced morphological alterations of the neurons, as well. Vacuolization of the cytoplasm in close correlation with desorganisation of the cytoskeletal elements were characteristic in this respect. Similarities and differences of neurotoxic actions of aluminum on invertebrate and vertebrate preparations are discussed.

P-18 THE ROLE OF IRON IN NEUROTOXICITY - A STUDY OF NOVEL ANTIMALARIAL DRUGS Smith, S.L., Maggs, J.L., Edwards, G.*, Ward, S.A., Park, B.K., McLean, W.G. Dept. of Pharmacology and Therapeutics, University of Liverpool, *Liverpool School of Tropical Medicine, Liverpool, U.K.

The antimalarial drug artemisinin and its derivatives display neurotoxicity in animal studies in vivo. Their antimalarial activity may be due to an interaction of the endoperoxide bridge of the derivative with iron. In this study, we have examined the role of iron in the neurotoxicity of artemisinin derivatives in vitro in the neuroblastoma NB2a cell line. NB2a cells in culture were induced to differentiate by removal of serum and addition of 0.5 mM dibutyryl cyclic AMP in the presence of 300 nM artemether or dihydroartemisinin with or without 2 mM haemin. After 24 h, cells were fixed and stained and neurite outgrowth quantified by image analysis. In some experiments, NB2a cells were incubated with 15 mM 14C-artemether and the haemin-induced conversion products in the medium were characterised by LCMS. Haemin significantly potentiated the neurotoxicity of artemether and dihydroartemisinin by 82% and 72%, respectively. In the case of dihydroartemisinin, the inhibition of neurite outgrowth produced by the drug alone was completely prevented by the cell permeable iron chelator CP40. Two major endoperoxide-bridge rearrangement products, viz. a compound preliminarily identified as 3a-hydroxydesoxyartemether and the tetrahydrofuran acetate isomer of artemether were formed in the presence, but not in the absence, of haemin. The results indicate the importance of iron in the neurotoxicity of artemisinin derivatives.

P-19 MORPHOLOGICAL CHANGES FOLLOWING CATECHOLAMINERGIC PC12-CELLS EXPOSURE TO MANGANESE COMPOUNDS, STUDIED BY CONFOCAL MICROSCOPY Vettori, M.V., Mutti, A., Gatti, R.*, Orlandini, G., Alinovi, R., Belletti, S.**, Smargiassi, A., Franchini, I. Dept. of Clinical Medicine, Nephrology and Health Sciences, Institutes of *Human Normal Anatomy and **General Pathology, Univ. of Parma Medical School, Parma, Italy

Mn is thought to cross the blood-brain barrier as a Mn-transferrin (Mn-Tf) complex, following internalization of Tf receptors expressed by endothelial cells. However, the physical state of Mn active in the brain is presently unknown. Both MnCl2 and manganic transferrin (Mn-Tf) were used in experiments with PC12 cells aimed at investigating Mn-induced morphological changes. Cells were seeded at a low density (4 · 104 cells/ml) in chambered coverglass used for studies by confocal laser microscopy. The vital fluorescent dye calcein was used to observe apoptosis, the FITC-Annexin V marking being employed as a confirmatory test. Cell necrosis was never seen in a broad dose interval (10-4-10-7 M) of both compounds. Treatment of PC12 cells with physiological and paraphysiological doses (10-5-10-7 M) was followed by time- and dose-dependent morphological changes characteristic of apoptosis, as observed by confocal microscopy. A significant apoptosis, defined as nuclear condensation or membrane blebbing in at least 10% of cells, was observed after 6 hours of exposure to MnCl2 and Mn-Tf 10-6 M; the highest concentration (10-5 M) induced morphological changes characteristic of apoptosis in a greater percentage of cells (about 35-40%). The translocation of phosphatidylserine (PS) from the inner side of the plasma membrane to the outer layer - an early signal of apoptosis preceding nuclear changes - was observed after 3 hours from the beginning of exposure to 10-5 M. Apoptosis can be prevented only in part by co-incubation with a 100-fold excess of ascorbic acid. This suggesting that the generation of oxygen radicals is not the main mechanism underlying Mn-induced programmed cell death. Acknowledgements : Supported by the European Union (ENV4-CT96-0173) and by a Québec IRSST scholarship.

P-20 THE LEVELS OF CADMIUM IN THE FOOD AND THE MATERNAL BODY, RISK FOR THE HEALTH Hura, Carmen, Rusu, Lidia Institute of Hygiene, Public Health, Health Services and Management, Medical Center, Iassy, Romania

Ever since humans have become aware that health is inseparably linked to an intact and healthy environment, the control and reduction of pollution have become the focus of world-wide concern. Cadmium occurs in nature in trace amounts. Nevertheless, because of its exceptional environmental persistence and ability to cumulate throughout almost whole life-span of humans, it is one of the most dangerous environmental contaminants. The study presents the results of cadmium concentrations in some food (milk, bread, daily diets) and maternal body (milk, placenta) during the years 1995-1996, in Moldavia (Romania). Atomic absorption spectro-photometry was used for the determination of cadmium contents in food and human body. The results of the investigations showed a significantly increased Cd content in milk and daily diets in Bacáu district with a mean level 1.34 mg/kg milk respectively 1.0 mg/kg daily diets. In other regions of Moldavia, Cd contents in milk varied between 0.7 mg/kg - Botosani district and 0.23 mg/kg - Galati district [MAC Cd for milk is 0.1 mg/kg]. Cadmium levels in the maternal body were generally low: mean 2.3 mg/g in human milk and mean 2.0 mg/g in placenta. These results stress the fact that there is a moderate change with cadmium in the population of our country, which can be considered as a result of chemical pollution.

P-21 VANADIUM AS A FACTOR THAT DISTURBS PHOSPHORUS METABOLISM IN NERVOUS TISSUE Parsadanian, H.K., Marchenko, S.N., Parsadanian, K.H., Barilyak, I.R., Ter-Tatevosian, L-P.* Ukrainian Scientific Center of Hygiene, Kiev, Ukraine, *Institute of Biochemistry of the Armenian NAS, Yerevan, Armenia

Vanadium and its derivatives are the well-known environmental pollutants. On the other hand this metal is an essential trace element with insufficiently explored biological function. We obtained that ammonium vanadate suppressed the alkaline (AlP) and acid (AP) phosphatases activity in a variety of tissues. Vanadate inhibited the enzymes that take part in phosphoryl transfer reactions. Successive concentration-dependent decrease of AlP and AP activities by vanadate (0.1 mM - 1 mM) has been obtained in rat nervous tissue. Moreover, the observed reduction of the sensitivity of the enzymes to vanadate ions becomes more pronounced at the transition from higher and relatively young regions of ner nervous system to its more ancient parts. Phosphoprotein phosphatase (PP) activity has also been inhibited in various structures of brain tissue; in spinal cord, however, vanadate in comparatively low concentrations (10 mM) caused a steep rise in the enzyme activity. Only at high concentration (1 mM) vanadate exert the effect of moderate inhibition. The possibility of the existence of phylogenetically different molecular forms of nervous tissue PPs characterized by their sensitivity to vanadate was supported in our comparative examination of two PPs: PP-1 and PP-2B. Several thyolic amino acids are supposed as possible protectors against vanadium effect.

P-22 THE EFFECT OF VANADATE UPON SEVERAL METABOLIC PROCESSES AND THE WAYS OF ITS NEUTRALIZATION IN NERVOUS TISSUE Parsadanian, K.H., Marchenko, S.N., Parsadanian, H.K., Ter-Tatevosian, L.P.* Ukrainian Scientific Centre of Hygiene, Kiev, Ukraine, *Institute of Biochemistry of the Armenian NAS, Yerevan, Armenia

Some effects of vanadate upon metabolic processes in rat nervous tissue have been demonstrated in part in our previous study (H.K.Parsadanian et.al. 1991). Concentration-dependent changes in glycogen phosphorylase (GP) activity observed are shown. to be in reciprocal correlation with changes taking place in protein phosphatase (PP) activity. At the same time, millimolar concentrations of vanadate inhibit GP in various parts of nervous tissue. The same concentrations of vanadate suppress also protein kinase activity in brain and spinal cord. Among the reagents used as potentiat protectors cysteine was the most effective factor that was able to keep PP activity in rat brain and the other tissues excepting that of the heart muscle.

P-23 EFFECTS OF TRIMETHYLTIN ON STARTLE RESPONSE AND BRAIN SIZE IN RAT PUPS Allen, S.L., Duffel, S.J., Jones, K., Soames, A.R. Zeneca Central Toxicology Laboratory, Macclesfield, U.K.

Groups of twenty, 8 day old, male and female Wistar-derived rats were dosed intraperitoneally with 0, or 8 mg trimethyltin chloride/kg using deionised water as a vehicle. Detailed clinical observations and body weights were recorded for all animals at regular intervals throughout the study. Startle response testing was performed on 10 rats/sex on day 23. Selected animals were terminated on days 12 and 24 post partum and histopathological examination of the brain, including morphometric analysis, was performed. At 8 mg trimethyltin chloride/kg significant reductions in body weight and changes in auditory startle response (reductions in amplitude and/or a delay in the time to peak effect) were seen in both male and female rat pups, generally in the absence of clinical signs of toxicity. Significant reductions in brain weight and histopathological lesions in the hippocampus and/or the pyriform and entorhinal cortex on both day 12 and 24 were also seen for these animals. These effects correlated with significant reductions in morphometric measurements (reductions in the size of cortical, thalamic and hippocampal regions of the brain). The results of this study confirm and extend the reported literature of the effects of trimethyltin chloride on startle response and brain size.

P-24 PHARMACOLOGICAL ACTIONS OF ESSENTIAL (ZINC, COPPER) AND TOXIC METAL IRONS (LEAD, MERCURY) ON NEURONS AND NEURONAL SYNAPSES OF THE SNAIL, HELIX POMATIA L. Erdélyi, L., Kovács, T., Insperger, K., Földesi, Zs. Attila József University of Sciences, Department of Comparative Physiology, Szeged, Hungary

Physiological metal ions are known as modulators of various neuronal events and both they deficiency and excess can cause functional disturbances ( Vallee and Falchuk, 1993). Some metal ions, like lead and mercury are toxic elements (Vallee and Ulner, 1972; Kiss and Osipenko, 1994). We studied the actions of the above mentioned essential and toxic metals on neuronal excitability of the snail, Helix pomatia L. The experiments were carried out on identified neurons under current- and voltage-clamp conditions. The active and passive membrane characteristics of the neurons and spontaneous or stimulus evoked excitatory postsynaptic potentials or currents ( EPSPs or EPSCs) were examined. The physiological solution contained (mM): NaCl, 80; KCl, 4; CaCl2, 7; MgCl2, 5; Tris-HCl, 5 at pH= 7.5. The L and RPa 2,3; RPa4; V1 and RPl1 neurons were used as identified cells. The metal ions were applied in the perfusate in uM or sometimes in mM concentrations. The Student, t-test was used for statistical evaluation of the results. Among the studied metal ions zinc (100-400 uM) and copper ( 10-150 uM) ion treatment increased the frequency of the spontaneous EPSPs or EPSCs most effectively. However a concomitant decrease in the amplitude of the elementary spontaneous events are developed. Low dose of Zn and Cu also increased the peak amplitude of the stimulus evoked EPSPs or EPSCs, but in higher dose they suppressed it in a dose-dependent way (IC50= 73 and 85 uM respectively). Zn in 1-2 mM and Pb in 100 uM concentrations sustained the voltage dependence of the evoked postsynaptic responses. Mercury ions ( 1-50 uM) transiently increased the frequency of the spontaneous and peak amplitude of the evoked responses but after a short delay the effect was irreversibly suppressive on all components of the synaptic events (IC50= 10 uM). The studied metal ions decreased the resting membrane potential, the amplitude of the action potentials and the input resistance of the neurons. In close correlation with the current clamp observations the studied metal ions increased the holding and leak currents in the voltage clamped cells. Especially zinc ions attenuated the fast outward current which in close correlation with the membrane and synaptic actions increased the neuronal excitability in the identified neurons. The actions of Cu, Pb and Hg ions are predominantly toxic on Helix neurons.

P-25 SERUM DEPRIVATION - ISHEMIA-INDUCED APOPTOSIS IS NOT SENSITIVE TO GLUTAMATE RECEPTOR ANTAGONISTS Kalda, A., Zharkovsky, A. Department of Pharmacology, University of Tartu, Estonia

In the present study, the mechanisms of death induced by transient oxygen-glucose deprivation (OGD) and the effect of serum deprivation after OGD in rat cerebellar granule cells were studied. The exposure of the cells to OGD (90 min) and consequent incubation in conditioned medium for 6-24 hours led to the necrosis (increase in the activity of lactate dehydrogenase, LDH) and apoptosis (increase in the number of positive the terminal deoxynucleotydil transferase-mediated dUTP-biotin nick end labelling-stained cells, TUNEL). Both, necrotic and apoptotic cell death were prevented by the addition of NMDA receptor antagonist MK-801 (l0 mM) and AMPA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, l00 mM). When OGD exposed cells were incubated with fresh medium lacking serum, MK-801 and CNQX also almost completely inhibited the rise in LDH activity, but increased the number of TUNEL positively stained cells. These data demonstrate that serum deprivation increases apoptosis after OGD that is not sensitive to glutamate receptor antagonists.

P-26 EXAMINATION OF 3H-BATRACHOTOXININ AND 3H-LIFARIZINE BINDING TO RAT BRAIN SODIUM-CHANNELS Tömösközi, Zsuzsanna, Baranyi, A., Békási, E., Pascal, M., Arányi, P. Chinoin Co. Ltd., Budapest, Hungary

Neurotoxins have highly specific actions on molecular targets, and thus offer an effective means of characterizing the growing number of identified ion channels and receptors in the nervous system. Highly selective neurotoxins proved to be useful tools for investigation of physiological, cellular and molecular mechanisms underlying brain fuction as well. Voltage-dependent sodium-channels (Na-channels) are multisubunit protein complexes in the membranes of the neurons and muscle cells and play fundamental role in the generation of electric stimuli. To date, more than five distinct neurotoxin-binding sites have been identified on them. Some of these neurotoxin-sites are associated with activation, inactivation and permeation properties of the channel. Batrachotoxin (BTX), a specific activator for these channels is a widely accepted probe for the demonstration of the activated channel-state. Lifarizine, a diphenylpiperazine analogue displaces BTX binding on its sites with a moderate affinity, indicating an interaction with the Na-channel. 3H-Lifarizine binds specifically to the activated channel but has higher affinity for the channel in its inactivated state. The aim of our study was to examine the interactions of 3H-BTX and 3H-Lifarizine with the recombinant rat brain type IIA Na-channels and with the native Na-channels in the rat brain.

P-27 EFFECT OF SUBUNIT COMPOSITION ON THE KINETIC PARAMETERS OF RAT CORTICAL SODIUM CURRENTS Gyõri, J., Baranyi, A., Jenei, A., Pascal, M., Arányi, P. Pharmacology of CNS, Chinoin Co. Ltd., Budapest, Hungary

Vertebrate brain sodium channels consist of an a and more ß subunits. The pharmacological profile of Na-channel subtypes is an important factor, which helps to determine the primary target of drugs. There are structural differences among subtypes, that correlate to important differences in the kinetic parameters as well. The aim of our study was to compare the inactivation properties of rat brain Na-channels, and transfected CHO cells expressing only athe type subunit. The kinetic parameters of voltage gated Na currents were analysed in whole cell patch clamp recordings on CHO cells, expressing the brain type CNA IIA Na+ -channel (obtained from W.A. Catterall, Univ. of Washington, Seattle, WA), and on rat primary cortical cell-culture. Cells were kept at -70 mV holding potential, and Na-channels were opened by 70 mV square pulse impulse lasting for 10 ms. Analysis of the inactivation parameters showed remarkable differences between cell types. In case of CHO cells the Na-currents inactivated very fast, and reached zero level immediately, while inactivation in pyramidal shaped cortical neurones containing a and ß subunits was slower, and we detected a still remaining component. Our results suggest, that the lack of ß subunit modifies electrophysiological properties of sodium currents but the role of ß subunits in regulating the function of Na-channels is not clear at the moment.

P-28 NORBORNAN IS THE IRREVERSIBLE LIGAND FOR CHLORIDE CHANNELS OF GABAa RECEPTORS Golovko, A.I., Sofronov, G.A., Kljuntina, T.V., Ivanov, M.B., Sviderskij, O.A. Military Medical Academy, St.-Petersburg, Russia

The convulsant norbornan [ekzo,cis-5,6-dichlor-2,2-dicyano-3,3-bis(trifluoromethyl)-norbornan] have a higher potency at the GABA-gated chloride ion channel protein of rat synaptic cortical membranes compared to that of tetramethylenedisulfotetramine, t-butylbicyclophosphorous ester and picrotoxin. Preincubation of tissue with norbornan, followed by extensive washing, resulted in a concentration-dependent reduction in the number of 3H-TBOB binding sites. The administration of norbornan to rats and mice resulted in the gradual increasing of convulsive activity. The animals died after 6 h. It was found the inhibition of 3H-TBOB binding in striatum and cerebellum of treated rats and in whole mouse brain. Picrotoxin in equitoxic doses did not alter the 3H-TBOB binding. These findings suggest that GABA-antagonist norbornan is the acylating agent for chloride channels of GABAa receptors.

P-29 NITRIC OXIDE SYNTHASE, AND OXIDATIVE STRESS INDUCED IN THE BRAIN FOLLOWING EXPOSURE TO ALUMINIUM Bondy, S.C., Guo-Ross, S.X. Center for Occupational and Environmental Health, Dept. of Community and Environmental Medicine, University of California, Irvine, CA, USA

Aluminum has been implicated in Alzheimer's Disease but this potential correlation remains controversial. Increased oxidative stress has been identified as a contributory factor in a wide range of neurological disorders including Alzheimer's disease, Parkinsonism, amyotropic lateral sclerosis, epilepsy and stroke. Therefore, the potential of aluminum salts to effect increased production of reactive oxygen species was of interest. While aluminum salts alone are not able to promote pro-oxidant events in isolated systems, they are able to potentiate the free-radical inducing properties of iron. Treatment of adult rats with aluminum over a two week period increased the rate of generation of reactive oxygen species in cerebral tissues while glutathione levels were also higher. These effects were not enhanced by a concomitant exposure to dietary iron. Levels of cerebellar nitric oxide synthase were also elevated in aluminum-treated animals. Apopain, an enzyme selectively induced in cells undergoing apoptosis, was specifically elevated following aluminum exposure. Thus, aluminum may promote pro-oxidant status and cell death within the brain, and an induction of nitric oxide synthase may underlie these events. Supported by NIH Grant # ES 7992.

P-30 SYNERGISM BETWEEN NMDA AND DOMOIC ACID IN A MURINE MODEL OF BEHAVIOURAL NEUROTOXICITY Tasker, R.A.R., Strain, S.M. Department of Anatomy and Physiology, Atlantic Veterinary College, University of Prince Edward Island, Charlottetown, PEI, Canada

Domoic acid (DOM) is generally believed to be a selective agonist for a subtype of AMPA/kainate excitatory amino acid receptors. However, recent evidence using either isolated cerebellar neurons (Novelli et al. Brain Res. 577: 41 1992; Berman et al. Proc.Soc. Neurosci. 25: 1586, 1995) or intact mice (Tasker et al. CJPP 74: 1047, 1996) have implied that N-methyl-d-aspartate (NMDA) receptors may also be partially involved in mediating domoic acid toxicity in these models. The current study examined behavioural neurotoxicity in mice following co-administration of EAA-selective ligands or controls. Female CD-1 mice (n=4 per group) were injected and monitored for both cumulative behavioural toxicity and latency to tremors and convulsions using procedures published elsewhere (Tasker et al. CJPP 69: 378, 1991). Co-administration of NMDA (40 mg/kg) significantly potentiated the toxic response to DOM (3.75 and 5.0 mg/kg) relative to saline/DOM controls but was without effect when administered alone at this dose (ie. NMDA/Sal) using either measure of toxicity. Further, NMDA potentiation was completely antagonized by prior administration of CPP (15 mg/kg). These data further support the premise that part of DOM-mediated neurotoxicity is dependent upon either direct or indirect NMDA-receptor activation. Supported by NSERC and the Atlantic Veterinary College.

P-31 HETEROGENEITY IN REGIONAL DISTRIBUTIONS OF GLUTATHIONE, ASCORBIC ACID AND ENDOGENOUS LIPID PEROXIDATION IN RAT BRAIN Hu, H.L., Bennett, N., Lister, T., Ray, D.E. Medical Research Council (MRC) Toxicology Unit, Leicester, U.K.

The regional distributions in brain of these two antioxidants, as well as the spontaneous lipid peroxidation have been assessed in F344 male rats by HPLC methods. GSH and ascorbic acid in brain have a very similar distribution pattern across brain regions. Their levels were lower in brain stem areas such as medulla; vestibular, cochlear, cerebellar roof nuclei; inferior and superior colliculi than the other brain areas such as striatum, hippocampus, neocortex, cerebellar cortex, and thalamus (p < 0.01). Striatum had the highest level of GSH (1.6 ±0.09 mmol/g) while hippocampus had the highest level of AA (2.7±0.17 mmo1/g). Medulla had the lowest level of GSH (0.97±0.10 mmol/g) whereas cochlear nuclei had the lowest level of AA (0.83±0.08 mmol/g), both areas are in brain stem. In contrast, the levels of free malondialdehyde (MDA), an index of oxidative stress, were much higher in the brain stem than in other areas, e.g. hippocampus, striatum, thalamus, cerebellar cortex, and neocortex (p< 0.01 ). Moreover, we have previously shown brain stem areas to be more sensitive to GSH depletion induced by L-buthionine-SR-sulfoximine (BSO) due to the faster GSH turnovers in those areas. These results suggest a lower antioxidant capacity and a higher spontaneous oxidative stress in the brain stem areas. This may partially explain the susceptibility of brain stem to neurotoxic xenobiotics such as 1,3-dinitrobenzene.

P-32 KAINIC ACID-INDUCED NEUROTOXICITY STUDIED ON HIPPOCAMPAL SLICES Ábrahám, H., Czeh, G.*, Lázár, Gy. Department of Human Anatomy, *Department of Pharmacology, Medical University of Pécs, Pécs, Hungary

Kainic acid is a glutamate analogue excitotoxin which induces neuronal degeneration in the hippocampus within a few days. The early functional changes of the neurons in the presence of kainic acid and the recovery of the cells after the exposure can be recorded using electrophysiological techniques. In the present study we have examined the early microglial response to kainic acid neurotoxicity. Brief administration of kainic acid into the recording chamber is a relatively easy procedure, therefore we used this test to study the early functional changes in neuronal activity and to obtain tissue for immunohistochemistry of glial cells. In order to study the glial activation after the electrophysiological recordings 40 mm frozen sections were made from the approximately 500 mm thick hippocampal slices. Using immunohistochemical method microglial cells were detected with a monoclonal antibody raised against the CR3 receptor (OX-42). Using the Neurolucida Image Analyser program we quantified the activated cells and their distribution in the kainic acid treated slices. Non activated microglial cells have appeared different morphology in control slices. Early microglial reaction could be observed by the increased OX-42 (CR3 receptor) immunoreactivity and the morphological changes after a 15 min kainic acid injury. Rapid activation of the cells suggests that the microglial reaction precedes the neuronal cell death and can be a good indicator of the toxic effects of excitatory amino acids in the central nervous system.

P-33 IN-VITRO EFFECT OF THE CYSTEINE METABOLITES HOMOCYSTEIC ACID, HOMOCYSTEINE AND CYSTEIC ACID UPON HUMAN NEURONAL CELL LINES Parsons, R.B., Waring, R.H., Ramsden, D.B.*, Williams, A.C.** Dept. of Biochemistry, Univ. of Birmingham, Edgbaston, Birmingham, *Dept. of Medicine and **Centre for Neurosciences, Univ. of Birmingham, Queen Elisabeth Hospital, Edgbaston, UK

Cysteine (CYS) is a non-essential amino acid which elicits excitotoxic properties via the N-methyl-D-aspartate (NMDA) subtype of the glutamate receptor. Many studies have described elevated CYS levels in association with many neurological diseases such as Parkinsons Disease (PD) and Alzheimers Disease (AD). Studies by this group and others have investigated the major catabolic enzyme of CYS, Cysteine Dioxygenase (CDO), which has been reported to have reduced activity leading to elevated CYS levels in the Globus Pallidus in the rare neurological disease Hallervordern-Spatz disease. We have previously reported studies investigating the toxicity of CYS and its major metabolite cysteinesulfinic acid (CSA) to human neuronal cell lines in vitro and in continuation of this we report the toxicity of other metabolites of CYS in the same system. Three cell lines of human origin (TE 671, U-87 MG, SK.N.SH derived from a medulloblastoma, glioblastoma and neuroblastoma respectively), were cultured in media containing various amounts of homocysteic acid (HCA), homocysteine (HCYS) or cysteic acid (CA). After the test period the media was assayed for lactate dehydrogenase release as a measure of cell death. The cell lines investigated showed varying degrees of toxic response which was the inverse to that seen when CYS or CSA was used. SK.N.SH, which exhibited a high toxic response to CYS or CSA exhibited a low toxic response to HCA, HCYS and CA with the converse being true for TE 671 which showed a low toxic response to CYS and CSA. The cell line U-87 MG, which exhibited a median toxic response to CYS and CSA, exhibited a similar median toxic response to HCYS, HCA and CA. These results show that toxic responses are cell specific for CYS and its metabolites. Neurodegeneration observed in neurological diseases such as PD and AD may therefore reflect the relative impact of CYS and its metabolites on the different cell types present in the brain. Defects in CDO have been noted in PD and AD and this may lead to failure to metabolise CYS to non-toxic products. It is interesting that HCYS is very toxic at low levels (2 - 10 µM) to the cells, particularly the TE 671 Medulloblastoma. This may be relevant to the HCYS toxicity which results in neural tube defects in prenatal humans.

P-34 EFFECTS OF KETAMINE ISOMERS ON THE GLUTAMATE-INDUCED NEUROTOXICITY AND 45CA2+ UPTAKE INTO PRIMARY CULTURE OF CEREBELLAR GRANULE CELLS Eriste, E., Kalda, A., Zharkovsky, A. Department of Pharmacology, University of Tartu, Estonia

Ketamine is non-competitive antagonist for N-methyl-D-aspartate (NMDA) receptors, and its anaesthetic, analgesic actions are mainly linked to this action. Recent studies have demonstrated that S(+)-isomer of ketamine is judged to produce more potent anaesthesia than either the racemate or R(-)-isomer. In the present experiments, the effect of S(+)- and R(-) ketamine on glutamate (500 mM)-induced toxicity and NMDA (100 mM)-evoked calcium uptake into primary cultures of cerebellar granule cells were studied. S- and R-ketamine in micromolar range of concentrations inhibited glutamate-induced neurotoxicity as revealed by lactate dehydrogenase and trypan blue assays. In neurotoxicity assays S-isomer was 10-30 fold more potent than R-isomer. Both S(+)-ketamine and R(-)-ketamine inhibited 45Ca2+ uptake in a concentration dependent manner with IC50 for S(+)- and R(-)- isomers, 5.4±2.0 and 39.7±14.0 nmol/mg protein/5 min, respectively. It might be proposed that higher pharmacological potency of S(+)-ketamine is related to its larger inhibitory action on the NMDA-linked calcium channel.

P-35 EFFECTS OF VITAMINS E AND C ON HYDROGEN PEROXIDE- AND tert-BUTYLHYDROPEROXIDE-INDUCED PRODUCTION OF REACTIVE OXIGEN SPECIES IN NEUROBLASTOMA CELLS Eerikäinen, S., Naarala, J., Savolainen, K. Department of Pharmacology and Toxicology, University of Kuopio, Kuopio, Finland

We show here that production of reactive oxygen species (ROS) could be decreased by very low concentrations of vitamins E and C in human neuroblastoma cells. The production of ROS was elicited by hydrogen peroxide (H2O2) or by a lipoperoxidative agent, tert-butylhydroperoxide (t-BOOH). The formation of ROS was measured using a fluorescent probe, dichlorofluorescin. Vitamin E was effective in decreasing ROS production elicited by H2O2 already at a 0.1 pM concentration. However, at 10 and 100 pM concentrations the block was only at 50% level. Higher concentrations of vitamin E blocked almost completely ROS production initiated by H2O2. Vitamin E also effectively decreased t-BOOH-induced ROS production at a concentration range of 0.1 pM - 1 mM. Vitamin C had also a biphasic effect on H2O2-induced ROS production, blocked by both low (0.1 µM) and high (100 µM - 1 mM) concentrations of vitamin C. At concentrations of 1 and 10 µM of vitamin C the ROS production stayed at a level caused by H2O2 alone. At a high concentrations (500 µM - 1 mM) vitamin C completely blocked t-BOOH-induced ROS production. It seems that both H2O2- and t-BOOH-induced ROS production is very sensitive to the effects of vitamin C and E. Vitamin E inhibits t-BOOH-induced ROS production more effectively than vitamin C. Supported by the Academy of Finland.

P-36 THE EFFECT OF DITHIOCARBAMATES ON DOPAMINE TRANSPORT SYSTEMS Bachurin, S.O., Petrova, L.N. Institute of Physiologically Active Compounds, Chernogolovka, Russia

Earlier it was shown (Bachurin et al., Neurotoxicology 1996, 17: 3-4) that number of dithiocarbamates (DTC) can potentiate neurotoxic properties of MPTP, which induces in human and in some laboratory animals a parkinsonism-like disorder by selective accumulation of its metabolite MPP+ in dopamine nerve terminals. In the present study we have analyzed the DTC effects on dopamine (DA) uptake and release systems in mice brain synaptosomes. It was revealed that at low concentrations (less than 100 mM) DTC weakly effect the [H3]MPP+ uptake into DA nerve terminals. The most pronounced inhibition of [H3]MPP+ uptake was observed for ethylcyclohexyl derivative (ECH-DTC). This compound also activate to some extent spontaneous release of [H3]DA and [H3]MPP+. Overall effects of DTC on the [H3]DA and [H3]MPP+ release is provided mainly via the activation of a K+-stimulated release process. Two compounds: Dicyclohexyl-DTC (DCH-DTC) and ECH-DTC strongly activated the K+-stimulated release of [H3]DA and [H3]MPP+ as well. However, at the lowest concentration DCH-DTC but not ECH-DTC inhibits the K+- stimulated release of [H3]DA. In the case of MPP+, this effect leads to the increase of neurotoxic metabolite content in the dopamine neurons. Thus, among 4 DTC derivatives studied, ECH-DTC has the lowest stimulatory effect on [H3]MPP+ accumulation in the DA-terminals, while DCH-DTC has the strongest effect. The observed regularities coincide well with the DTC neuropromotive activity in vivo.

P-37 ROLE OF IMMUNE SYSTEM IN DELAYED NEYROPATHY PATHOGENESIS Kokshareva, N.V., Zhminko, P.G., Shushurina, N.A., Kagan, Y.S., Hilkevich, T.V. Institute of Health, Kiev, Ukraine

The mechanism of the delayed neuropathy (OPIDP) (development of ataxia, paralyses and produce axon demyelinization in 14-21 days after exposure of some organophosphorus (OP) compounds) is not clear. Our investigation had shown that strong (at 70-90% ) neuropathy esterase inhibition in animals brain (rat, guinea-pig, hens) in 24 hours after treatment of TOCP, aphos, oxyphosphonate in toxic doses (0.5-LD50) is early indication of OPIDP. Electrophysiological investigation of the functional state of hen's peripheral nervous system revealed that conduction velocity decrease along of nerves was an preclinical sign of OPIDP development. For hens with OPIDP clinical picture manifestation of signs correlated with the degree of microdispersive circulating immune complexes accumulation, autoantibodies to tissue antigen from brain and with T-suppressers and NK-cells, which testified to autoimmune disorders under neuroparalytic OPIDP effect. The inhibition of nonspecific enzymes activity (cholynesterase, carboxylesterase) in nervous tissue are not specific and cannot be used for prognostication of OPIDP.

P-38 PECULIARITIES OF NEURO-MUSCULAR SYNAPSES OF MOUSE NECK MUSCLE AND EFFECTS OF ALLATOSTATIN I Surova, N.V., Balezina, O.P. Dept. of Human and Animal Physiology, Faculty of Biology, Moscow State University, Moscow, Russia

The neuro-muscular synapses of mammalian axial muscles are of special interest because of the complex pattern of innervation and anatomical structure of this musculature (Balezina, Poskonova 1991). Our histochemical studies of m.biventer cervicis synapses revealed the high percent of motor nerve terminal sprouting. The presence of 2 or more synaptic inputs per fiber are found in 24-35% of fast (white) neck muscle fibers. The evoked postsynaptic endplate currents (EPC) and potentials (EPP) demonstrate a high range of amplitude variations in the course of repetitive synaptic activity (4-50Hz). The fluctuations of EPP can reach 15-24 % from the mean value. In several muscle fibers the EPPs variations appear to have regular wave-form character with a constant period. The theoretical model was elaborated, predicting the EPPs amplitude changing, as a result of activity of two or more synaptic inputs coexisting in the muscle fiber, cointeracting or working independently. The data of model as well as the real EPPs fluctuations were also analysed using cross-, autocorrelation and Fourje analysis of EPPs variations. We conclude, that the nerve terminal sprouting and multiple innervation may play role in the postsynaptic activity of muscle fibers in neck muscles. The interactions of different synaptic inputs at the postsynaptic level may be the reason and physiological mechanism, determining the specific pattern of EPPs fluctuations in the course of prolonged repetitive activity. In the present study we also investigated the synaptic effects of insect neuropeptide allatostatin I (10-6-10-9 M). Experiments were carried out using the standard microelectrode technique, normal Liley solution and Mg2+- and d-tubocurarine blocks. The registration of miniature end-plate potentials (MEPP) and evoked end-plate potentials (EPP) was performed both on different synapses before/after substance application and on the same synapses before/after this application. We have achieved the following results: membrane potential seems to rise by 5 mv, MEPP amplitude increased by 30-100%, frequency of miniature end-plate potentials increased but not statistically significant. Also we saw amplification of EPP amplitude. Our data are not statistically significant.

P-39 SOME BEHAVIOURAL DISORDERS CAUSED BY L-ARGININE AND ITS ANALOGUES Savelieva, K.V., Kamensky, A.A. Dept. of Human and Animal Physiology, Faculty of Biology, Moscow State University, Moscow, Russia

As it is known L-arginine (L-Arg) is the endogenous precursor of recently discovered second messenger nitric oxide (NO). Although NO participates in normal synaptic transmission, excess levels of NO are neurotoxic. Previously we have shown the increase of NO level in the rats forebrain cortex 30 min after oral injection of L-Arg. The aim of this study was to investigate if oral administration of L-Arg in the range of doses might produce some disfunctions in rat's behaviour. We used the oral method of administration because it is the most physiological way for amino acids to get into the organism. It has been shown that L-Arg in doses of 50, 100 and 250 mg/kg impaired place learning with food reinforcement in the T-maze when injected 30 min before or immediately after learning sessions. We have shown that L-Arg injected 30 min before testing in the same doses increased the exploratory behaviour in rats which were habituated to the experimental equipment, compared to the control habituated rats. The doses of 5 and 500 mg/kg were ineffective. L-Arg in the dose of 250 mg/kg decreased the time of rats keeping on the Rota-Rod equipment. The analogues of L-Arg which are able to block the NO synthesis (NGnitro-L-arginine methyl ester and NGnitro-L-arginine) produced the effects opposite to the L-Arg effects. D-arginine in the same doses did not affect animal behaviour. So we may conclude that the excess amount of L-Arg may cause some behavioural disorders. First, it has a bad effect on animals memory in the maze task. Second, after L-Arg administration animals could not remember the experimental box. So they exhibit interest as if they were not habituated. Third, it may affect vestibular system of rats because L-Arg pre-treated rats endure rotation worse then control animals. Presumably, L-Arg realises its action through NO synthesis in the brain because the analogues of L-Arg induced the opposite effects and D-arginine failed to affect rats behaviour. The oral way of administration seems to us very perspectivic from the point of view for the therapeutic use of L-Arg and its analogues. The data obtained show the possibility to change or correct some behavioural parameters by L-Arg and its analogues increase or decrease in food products.

P-40 TOXICITY OF THE CYLINDROSPERMOPSIN PRODUCED BY CYANOBACTERIA CYLINDROSPERMOPSIS RACIBORSKII Hiripi, L., Nagy, L., Kovács, A., Vörös, L. Balaton Limnological Res. Institute of the Hungarian Academy of Sciences, Tihany, Hungary

Many drinking water supplies throughout the word experience periodic massive surface accumulation of cyanobacteria. Mass development of blue-greens in Lake Balaton became a regular phenomenon since 1973. The dominant blue-green alga since 1982 has been the tropical species Cylindrospermopsis raciborskii. Practically the whole lake was a mass culture of the toxic strain of this organism during the summer of 1994. Therefore, the aim of present study was to investigate the toxicity of the C.raciborskii. The alga was isolated from the lake, and subsequently cultured. To study the toxicity of the blooms and the cultured alga, we developed an insect test. The injection of the pure toxins (cylindrospermopsin, microcystin) or the extract of the cultured srains (C. raciborskii, Microcystis aeruginosa, Aphanizomenon flos-aquae) into the body cavity of the locust (Locusta migratoria migratorioides R.F.) led to the death of the animals. The sensitivity of the test was equivalent with the mouse test and was suitable to monitor the toxicity of the algal samples collected from the Lake Balaton. Using HPLC method the cylindrospermopsin was isolated from the cultured C. raciborskii. The effect of both algal extract and the pure toxin was investigated in fish, Rutilus rutilus L. Sublethal and lethal doses of the toxin caused liver demage and inhibited the respiration of the animal. This work was supported by a the Ministry for Enviroment and Regional Policy, The Balaton Secreteriat of the Office of the Hungarian Prime Minister and the Hungarian Scientific Research Fund (OTKA) Grant, No 22988.

P-41 MEMBRANE EFFECTS OF ALGAL TOXINS ON IDENTIFIED SNAILS NEURONS Vehovszky, Ágnes, Kiss, T., Kovács, A., Hiripi, L., Vörös, L. Balaton Limnological Research Institute of the Hungarian Academy of Sciences, Tihany, Hungary

The electrical responses of identified neurons of two snail species (Helix pomatia and Lymanea stagnalis) were studied using intracellular microelectrophysiological methods. Anatoxin-a had direct (depolarizing or hyperpolarizing) membrane effect on neurons, moreover reversibly blocked the acetylcholine responses. The cylindrospermopsin-containing exctract obtained from the pure Cylindrospemopsis raciborskii culture evoked similar membrane responses on identified neurons, and the acetylcholine responses of the neurons were also inhibited. The hepatotoxin microcystin however had no visible membrane effect recorded on snail neurons. Our results suggest that algal toxins have specific membrane effects on snail neurons and neurotransmitter receptors are also modulated both by anatoxin-a and the algal extract. Although cylindrospermopsin is considered as a hepatotoxin in Vertebrates (Ohtani et al 1992), the extract obtained from Cylindrospemopsis raciborskii culture also has clear neurotoxic effects on snail neurons.

Reference: Ohtani, I, Moore, R. E., Runnegar, M. T.: Cylindrospermopsin: a potent hepatotoxin from the blue-green alga Cylindrospermopsis reciborskii. J. Am. Chem. Soc. 114, 7941-7942. (1992).

This work was supported by a the Ministry for Enviroment and Regional Policy, The Balaton Secreteriat of the Office of the Hungarian Prime Minister and the Hungarian Scientific Research Fund (OTKA) Grant, No 22988.

P-42 FACILITATION OF SOCIAL RECOGNITION IN RATS BY COGNITIVE ENCHANCERS Bence, Judit, Baranyi, A. Pharmacology of Central Nervous System, CHINOIN Co.Ltd., Budapest, Hungary

The social olfactory recognition is employed to demonstrate the effects of compounds on short term memory and has been shown to be sensitive to cholinomimetics. The test is based on the fact that an adult male rat spends more time in investigating a novel, rather than a familiar conspecific juvenile. When the same juvenile is presented twice with relatively short interexposure interval, the duration of investigation by the adult is diminished on the second encounter. This reduced exploratory activity is considered as recognition of the conspecific. An unfamiliar juvenile rat was placed in the home cage of an adult rat for 5 min. The time spent by the adult in investigating the juvenile (close following, head-, body- and anogenital sniffing, grooming) was recorded. The adult rats were treated i.p immediately after the first exposure with vehicle or test compounds and 30 or 120 min later the same or a novel juvenile was placed in the home cage of the adult rat for a second exposure period of 5 minutes. Vehicle-treated adult animals investigated the same juvenile rat less than the novel one when the interexposure time was 30 min, while at 120 min rats investigated the same and novel juvenile equally, indicating that social recognition is not present any more. However, the AChE inhibitor Tacrine, the nootropic agent Piracetam, the M1 receptor agonist SR-46559A and a prolyl-endopeptidase inhibitor JTP-4819 facilitated the short term memory, as adult rats treated with these potential cognitive enhancers showed significant social recognition even after an interexposure interval of 120 min.. The results suggest that these drugs improve the short-term memory-related processes involved in social recognition of rats.

P-43 DERMORPHIN AND ITS NEW SYNTHETIC ANALOG INFLUENCE THE ANXIETY AND THE LEARNING PROCESS IN WHITE RATS Uranova, Maria G., Yarova, E.P., Deigin, V.I., Kamensky, A.A. Dept. of Physiology, Faculty of Biology, Moscow State University, Moscow, Russia

The neurotropic activity of dermorphin (DM) and its new synthetic analog, opilong-2 (Tyr-dAla-Phe-dAla-Tyr-Pro-Ser-NH-CH3) was examined in the elevated plus-maze paradigm and during acquisition of active avoidance task. The peptides were injected intranasally in dose 50 mg/kg 5 min prior to testing. Behaviours recorded in plus-maze test comprised the traditional indices of anxiety as well as a number of ethologically derived measures. A 5 min test duration was employed. The elevated plus-maze model is based upon an unconditioned aversion to heights and open spaces. Results show that these opioid peptides produced anxiolytic effect in animals. Rats treated with DM spent 72,8 sec in the open arms against 25,9 sec spent by control animals (p<0.005). DM and opilong-2 significantly increased total rears, total head-dipping and number of arm entries. The present findings demonstrate that the investigated peptides caused the anxiety reduction. We have also shown that the pre-training administration of DM and its analog inhibited acquisition of active avoidance task disrupting the process of forming the conditioned reaction. Therefore, these opioid peptides produce a wide range of effects on animals behaviour reducing the anxiety level and causing the amnesiac effect.

P-44 NEUROTOXICOLOGICAL INVESTIGATIONS OF CYTOSTATIC DRUGS IN ANIMAL EXPERIMENTS Ábrahám, S., Kiss, I., Borsa, M.*, Uray, Z.**, Sandu, V.D.*, Puica, C.*, Lenart, G. University of Veszprém, Dept. of Biology, Veszprém, Hungary, *Biological Research Institute, **Oncological Institute, Cluj-Napoca, Romania

Cytotoxic chemotherapy and radiotherapy have been mainstays in the treatment of cancer. The promise these therapies held the eradication of inoperable and disseminated malignancies. The administration of cytostatic drugs is often associated with significant organic and functional disorders in survivals. Our studies revealed that some anticancer therapy (chemotherapy and radiotherapy) may induce neuro-endocrine dysfunctions in laboratory rodents. Whole-body irradiation and/or administration of Cyclophosphamide, Cisplatin and Hydroxyurea affect the cellular structures and the enzymatic processes of the rat cortex, hippocampus, hypothalamus nuclei and of some endocrine glands. Using both histoenzymatical and biochemical methods we have studied the activity of acetyl- and butyrylcholinesterases, (K,Na)-ATP-ase, Ca-ATPase, Mg-ATPase, alkaline phosphatase and the activity of some oxido-reductases in the rat brain regions. The results obtained showed that especially Mg-dependent enzymes and cholinester hydrolases were affected strikingly in some brain regions after irradiation and/or cytostatic treatment. The animals irradiated and/or treated with Cyclophosphamide (40 mg/kg/day), Cisplatin (5 mg/kg/day) or other cytostatics suffered in severe neurological and metabolic disorders and often died within 10 days. These drugs caused enzymatic dysfunctions especially in the cortex, thalamus, hypothalamus and choroid plexus, which manifested by the inhibition of some enzyme activities and the diminution of intracellular enzyme populations in some brain regions.

P-45 SYMPATHETIC-ADRENAL SYSTEM (SAS) PROMOTES A TOXIC EFFECT OF 5-FLUOROURACIL ON HEMATOPOIESIS Gol'dberg, E.D.*, Dygai, A.M., Khloussov, I.A. Institute of Pharmacology, *Scientific Centre, Russian Academy of Medical Sciences, Tomsk, Russia

Administration of 5-fluorouracil (5-FU) in a high dose has been well known to cause the direct damages of hematopoietic cells and mature elements of blood system. We have established under 5-FU injection to mice that the cytostatic's toxicity is dependent on vegetative reaction on the irritant. Of interest is the fact, that 5-FU injection in a dose of 114 mg/kg diminished to a large degree catecholamines concentration (on 124th experimental days) in the adrenal glands examined by Hillarp, Hokfelt's method. In vitro adrenergic agonists had a weak stimulating (CFU-GM) or marked inhibiting (CFU-E) effects on the growth of hemopoietic precursors which were cultured on the 3d day after 5-FU injection. In turn, the double (in 5 hours) injection to mice of alpha- (dihydroergotaminum, 3.9 mg/kg) or beta-adrenergic antagonist (propranolol, 5 mg/kg) on day 3 afte r 5-FU administration led to a restriction of cytostatic's toxic influence on cells according to micronuclear test and accelerated a recovery of bone marrow indices. Thus, experiments allow to resume that close connections of SAS and blood system may enhance a toxic effect of 5-FU on hemopoiesis by means of negative catecholamines influence on cells damaged by the irritant.

P-46 MECHANISTIC INVESTIGATIONS CONCERNING THE FUNCTIONAL CNS IMPAIRMENTS OF THE NOVEL CYCLOSPORINE DERIVATIVE PSC 833 Cruz, F., Oberer, L., Cordier, A., Wolf, A. Novartis Pharma AG, Toxicology/Pathology, Basel, Switzerland

PSC 833 is a novel non-immunosuppressive cyclosporine, which was successfully used to reverse multidrug resistance during chemotherapy in clinical trials. In rats, PCS 833 causes transient neurological symptoms, such as ataxia, impaired locomotion, and hunched posture, while at similar dose-levels, cyclosporine A (CsA) is devoid of such effects. The mechanisms leading to these adverse effects are only poorly understood. Impaired glucose metabolism has been reported to be associated in various neurological disorders. It was therefore the goal of the present experiments to investigate if PSC 833 affects glucose metabolism in two in vitro brain models, primary cultures of rat cortical neurons and astrocytes. 13C-NMR was used as a tool to investigate the intracellular metabolic profile after incubation with (1-13C) glucose. The rate of glucose utilization and lactate production was spectrophotometrically determined in the medium. In neuron and astrocyte cultures, glutamate, glutamin, N-acetyl aspartate, alanin and lactate were clearly identified by their characteristic 13C resonances. Incubation with 10 mM PSC 833, a noncytotoxic concentration, for 24 hours caused a clear decrease in the Krebs cycle intermediates, reflected by glutamate and glutamin, which are precursors or by itself important neurotransmitters of the CNS, and increased intracellular lactate concentrations. In the medium, PSC 833 resulted an increased time-dependent and maximum 2 fold increased glucose utilization and increased lactate formation compared to controls. The results obtained with CsA under the same conditions differ only marginally as compared to controls, with the exception of decreased glutamin and glutamate concentrations, which were observed in neurons and astrocytes at cytotoxic concentrations of 10 mM. The present results suggest that PSC 833 causes an impairment in the Krebs cycle, which results in altered glucose metabolism and therefore acidosis. Both, the decrease in the Krebs cycle intermediates and the acidosis might be associated with the CNS side effects of the drug.

P-47 THERAPY FOR TYPE II PYRETHROID INSECTIDE POISONING Ray, D.E., Forshaw, P.J. Medical Research Council (MRC) Toxicology Unit, Hodgkin Building, Leicester, U.K.

Anticonvulsants are of limited value in controlling the acute excitation seen during human or experimental pyrethroid intoxication. We have shown that type II pyrethroids, in addition to prolonging the membrane sodium current, also reduce voltage-gated chloride current. This action may provide a theraputic opportunity. We evaluated two agents as antagonists of the action of the type II pyrethroid deltamethrin in male F344 rats. The agents were ivermectin (4 mg/kg i.v.), which increases chloride currents but has few central actions; and pentobarbitone (15 mg/kg i.p.) which acts centrally, although as a membrane stabiliser as well as on chloride channels. Phenobarbitone, a membrane stabiliser with little effect on chloride channels, was used as a pentobarbitone control at an equi-sedative dose (45 mg/kg). Agents were given prior to 1.5 or 2 mg/kg deltamethrin i.v. and the severity of choreoathetosis assessed as a score of 1-4. Ivermectin had only a small central effect, reducing the the 2 mg/kg choreoathetosis score from 3.9±0.1 (mean±S.E.) to 3.2±0.03 (n=16; p=0.023, Wilcoxon test); but was effective peripherally, reducing the incidence of salivation by 72%, and the amplitude of the deltamethrin generated electomyogram after discharge by 52% (p<.01; t-test). Pentobarbitone was effective centrally, reducing the 1.5 kg/kg choreoathetosis score from 3.0±0.4 to 1.3±0.03 (p=.004). The equisedative phenobarbitone control produced only a non-significant fall to 2.4±0.2 (p=.11). Thus agents acting to restore chloride conductance provide prophylaxis against both the central and peripheral effects of deltamethrin, and action on chloride flux may explain the anomalous efficacy of the barbiturate pentobarbitone.

P-48 STUDY OF THE DELAYED NEUROTOXIC EFFECT OF ORGANOPHOSPHORUS + PYRETHROID COMBINATION Vekovshinina, S.V. Institute of Health, Kiev, Ukraine

A number of organophosphorus (OP) such as mipafox, tryorthocresyl phosphate (TOCP), phosvel and others are known to produce the delayed neurotoxic effect (DNE). This effect is not established for synthetic pyrethroids (SP). But SP can strengthen of the neurotoxic effect of OP. In the study on hens, designed to determine whether deltamethrin (SP) potentiated the neurotoxic effect of classic neuropathic agent TOCP, 2 groups were given oral dose of TOCP (100 mg/kg) or a mixture of TOCP (100 mg/kg) and deltamethrin (15 mg/kg). The electrophysiological and Johnson methods were used. Following administration of mixture the signs were found similar to that observed during TOCP administration alone - ataxia and conduction velocity in hens (nervus adductor) at 14 and 21 days was not considerably different from control level. The activity of neurotoxic esterase - 40 % to control in the hens' brain following administration of TOCP + deltamethrin and TOCP was the same. The results showed that the synthetic pyrethroid, deltamethrin, did not increase the delayed neurotoxic effect of TOCP.

P-49 COMPARISION THE IMMUNO- AND NEUROTOXICOLOGICAL EFFECTS OF REPEATED SMALL DOSES OF AN ORGANOPHOSPHATE PESTICIDE, DDVP, IN THREE GENERATIONS OF RATS Institóris, L., Nagymajtényi, L., Siroki, Olga, Dési, I. Department of Public Health, Albert Szent-Györgyi Medical University, Szeged, Hungary

Repeated 1/50, 1/75, and 1/100 x LD50 doses (1.94, 1.29, and 0.972 mg/kg respectively) of DDVP were administered orally to Wistar rats throughout three consecutive generations including pregnancy and lactation. General toxicological (body weight gain, the weight of 9 organs), haematological (absolute and differential WBC, RBC, Ht, mean volume of RBCs, bone marrow cellularity), and immune function (PFC number of the spleen, DTH reaction) parameters in 8 weeks old males of each generation were determined. Paralelly, from separate groups of animals, neurotoxicological parameters (ECoG, somatosensory, visual, and auditory cortical evoked potentials, conduction velocity and refractory periods of peripheral nerve) were recorded at the age of 9 weeks. The cholinesterase activity of the brain was also measured. Among the general toxicological parameters the relative liver weight increased significantly in the second, and the relative thymus weight in the third generation. A decrease in some haematological parameters Ht, MCV, and bone marrow cellularity) was also observed in the second generation, in the third one these changes became more characteristic. The PFC content of the spleen decreased in the first generation only at the top dose, in the second one at the middle and top doses, in the third one at all the three doses. Among the neurological parameters a significant decrease in the ECoG index, and an increase of the absolute and relative refracter periods were found at all doses in all the three generations. The applied doses had no significant effect on the cholinesterase activity of the brain. On the basis of these findings the ECoG index and the refracter periods of the periferal nerve proved to be the most sensitive parameters in the applied system. For early detection of DDVP toxicity the neurotoxicological approach was more sensitive than the immunotoxicological one in all the three generations.

P-50 SELECTION OF STRATEGIES - AND CHOLINERGIC TASK MEDIATION - IN PLACE LEARNING OF THE RAT: THE SIGNIFICANCE OF DISTAL CUES Beltoft, V., Simonsen, L., Lund, S.P., Hass, U., Mogensen, J.* National Institute of Occupational Health, Lab. of Neuropsychiatry, *Dept. of Pharmacology, University of Copenhagen, Copenhagen, Denmark

Place learning of rats in water mazes may be divided into types: e.g. the "mapping type" and the "non-mapping type". "Mapping type" is seen when the rat is offered a free view of a room in which numerous cues are present. The mediation of the "mapping type" receives significant contributions from the cholinergic system. The mediation of the "non-mapping type" involves only insignificant contributions from cholinergic mechanisms. "Non-mapping type" has been observed when a curtain along the edge of the maze blocked the view of the room. Restriction of the cues available in the room may be sufficient to cause the "non-mapping type" to become the perfect strategy. Consequently, it may be hypothesized that restriction of the number of cues available in the room may be sufficient to cause a reduction of the degree to which the task is mediated by cholinergic mechanisms. To test this hypothesis we studied - in two separate experiments - groups of rats given i.p. injections of either scopolamine (1 mg/kg) or saline 20 min prior to testing. In both experiments the acquisition of a place learning task in a water maze was studied. In Exp. 1 relative few distal "cues" were present. In Exp. 2 several cues had been added to the room. In Exp. 1 the task acquisition failed to reveal significant group differences, while in Exp. 2 the task acquisition of scopolamine treated animals was significantly impaired. Even when a free view of the experimental room is offered, restriction of the number of distal cues may be associated with a "shift" towards selection of "non-mapping type" place learning - and consequently a reduced cholinergic contribution to the task mediation.

P-51 NEUROBEHAVIORAL EFFECTS OF MPTP IN C57BL MICE: A COMPARISON OF DIFFERENT TREATMENT REGIMES Gilbert, S.G., Charleston, J.S.* Institute of Neurotoxicology and Neurological Disorders, *Department of Environmental Health, University of Washington, Seattle, WA, USA

While several studies have shown that MPTP-treated C57BL mice are suitable models for Parkinsons disease there has been no systematic comparison of the onset and recovery of the neurobehavioral effects following different dosing regimes. In the present study, C57BL mice (6 per group) were given IP injections of MPTP as follows: a) 2 doses of 30 mg/kg at 6 hr intervals for 5 days; b) 4 doses of 20 mg/kg at 2 hr. intervals for 1 day; c) 3 doses of 20 mg/kg at 3 hr. intervals for 2 days; d) 2 doses of 20 mg/kg at 3 hr. intervals for 5 days; e) 4 doses of 2CH3-MPTP at 10 mg/kg at 2 hr. intervals for 1 day; f) 2 doses of MPTP at 36 mg/kg at 16 hr.. with Acetaldehyde (250 mg/kg) 10 min before and 10 and 30 min after the injection of MPTP; and g) a control group received saline injections at least twice per day. The neurobehavioral effects were examined using the Functional Observational Battery (FOB), a well characterized rodent behavioral screening procedure that includes assessment of activity, autonomic, neuromuscular and sensory system function. FOB assessments were done prior to dosing, the next day after the end of dosing and twice a week there after. Additional activity monitoring was done by assessing grid crossing over a 1 min period. Morphometric evaluations were done on the Substantia Nigra Reticulata (SNR) and the Substantia Nigra Compacta (SNC) neurons using the recently developed Optical Volume Fractionator (OVF) methodology (Bolender and Charleston, 1993). of the control, Group 1 MPTP (30 mg/kg) and Group 5 CH3-MPTP. Only the 2CH3-MPTP showed significant signs of reduced motor activity. There was a significant reduction in neuronal cells in the SNC in both groups compared to the controls but not in the SNR region. While acute effects of MPTP could be readily observed, overall the FOB was not sensitive to the predicted effects of MPTP neurotoxicity.

P-52 EFFECT OF AN ACUTE TREATMENT OF QUINUCLIDINYL BENZILATE ON MUSCARINIC ACETYLCHOLINE RECEPTORS (MChR) AND ACETYLCHOLIN ESTERASE (AChE) IN THE RAT BRAIN Malygin, V., Serebryakova, Olga, Makhaeva, Galina Institute of Physiologically Active Substances, Russian Academy of Sciences, Chernogolovka, Russia

The cholinergic neurotransmission in a brain of adult Vistar rats was disrupted by a single injection of cholinolytic quinuclidinyl benzilate (QNB, 0.1mg/kg, i/p). In different times (1-12 days) after the single injection the rats were decapitated an a specific binding of [3H]QNB to MChR as well as catalytic properties of AChE of the rat brain were examined. The acute administration of QNB caused an increase both in the affinity of MChR (the Kd values decreased from 0.073±0.005 to 0.032±0.004 nM; the 3rd day after dosing) and in their density (Bmax). The Bmax values increased from 1.217±0.047 (control) to 1.521±0.031 pmol/mg pr., the 3rd day). The binding properties of MChR returned to control value at the 12th day after acute injection of QNB. The decrease of AChE catalytic properties after the acute QNB treatment was also exhibited. The Km value of AChE changed from (1.41±0.19)x10-4 (control) to (2.57±0.21)x10-4M in QNB treated rats. Vmax values of AChE decreased from 1.45±0.21 (control) to 1.09±0.11 unit/min/mg protein. In summary, the acute injection of strong cholinolytic QNB results in the significant, long-term (up to 12 days) reversible increase in sensitivity of MChRs and the decrease in catalytic properties of AChE. These changes have apparently an adaptive character and could be used as the biochemical markers for Alzheimer's disease diagnostics. This research is supported by RFBR (Grant 96-04-50252)

P-53 COMPARATIVE STUDIES OF O,O-DIALKYL-O-CHLOROMETHYLCHLORO-FORMIMINO PHOSPHATES INTERACTION WITH NEUROPATHY TARGET ESTERASE AND ACETYLCHOLINESTERASE Makhaeva, G.F., Filonenko, I.V., Fomicheva, S.B., Malygin, V.V. Institute of Physiologically Active Substances, Russian Academy of Sciences, Chernogolovka, Russia

Neuropathy target esterase (NTE) is a molecular target for organophosphates (OP) which induce delayed neurotoxicity (OPIDN). The relative potency of an OP to react with AChE or with NTE in vitro correlates with its potency in vivo: death or OPIDN. On this basis extrapolation from in vitro to in vivo data now seems justifiable to predict risk of OPIDN. The kinetics of NTE and AChE inhibition by experimental pesticides (RO)2P(O)ON=CClCH2Cl has been studied. The obtained in our lab stable lyophilize preparation of paraoxon-pretreated (P2+P3) membrane fraction of hen brain homogenate was used as a source of NTE.

R Me Et i-Pr Pr i-Bu Bu Am lgkII(NTE) 2.33 3.30 2.65 4.12 5.04 5.43 6.17 lgkII(AChE) 4.96 4.83 3.85 5.25 5.90 6.06 5.91 kII(NTE) / kII(AChE) 0.002 0.03 0.063 0.075 0.139 0.230 1.83

Compounds with R = Me ( Pr are far more potent inhibitors of AChE than NTE. Both antiNTE activity and selectivity to NTE and, correspondently, risk of OPIDN rise with the increase of hydrophobicity. High values of kII(NTE) / kII(AChE) for compounds w th R > Pr indicate to their potential OPIDN in doses lower than LD50. The performed QSAR analysis demonstrated the different requirements of two the main OP target enzymes to the structure of OP inhibitors. The study was partly supported by Grant of RFBR No 96-04-50252.

P-54 EFFECTS OF TYPE II PYRETHROID DELTAMETHRIN ON THE CYTOSKELETON OF CORTICAL ASTROCYTES Lehmann, K.J., Grund, S., Stoltenburg-Didinger, G. Department of Neuropathology, Free University Berlin, Germany

Deltamethrin is one of the most widely distributed pyrethroids. The target site of pyrethroids are the sodium channels of the nerve membrane. To detect neurotoxicity in mammals, adult female Wistar rats were exposed to deltamethrin by a single intraperitoneal injection of two different concentrations (7.5 mg and 15 mg/kg body weight). Sham-injected and unexposed animals served as controls. In awake animals EEG was recorded weekly for 13 weeks. After the end of the experiment, animals were killed. Coronal paraffin sections were stained with antibodies to glial fibrillary acidic protein (GFAP), vimentin and S 100 protein. There was a dose-dependent increase of GFAP activity in astrocytes throughout the cortex but neither hyperplasia nor hypertrophy nor neuronal damage could be observed. In the cerebellum GFAP was highest in the highest dose group and in the roof of the fourth ventricle. Vimentin and S 100 reaction was unchanged. The basic mechanism of deltamethrin, the opening of neuronal sodium channels, leads to an increase of extracellular potassium. Increased concentrations of potassium are known to induce GFAP synthesis in astrocytes. As GFAP increases in a dose-dependent manner after deltamethrin exposure, the elevation of the extracellular potassium could be the link between the increased GFAP expression and the action of deltamethrin.

P-55 IN UTERO EXPOSURE TO LOW LEAD LEVEL-INDUCED NEURO-DEVELOPMENTAL EFFECTS ON INFANTS OF 9 MONTHS: INVOLVEMENT OF MONOAMINE NEUROTRANSMITTERS Tang, H.W., Huel G., Campagna, D., Hellier, G., Boissinot, C., Blot, P. Section of Environmental Toxicology, Unit of Epidemological Research, French National Institute of Medical Research and Health, Paris, France

To determine the possible involvement of dopaminergic and serotoninergic neurotransmitters in lead induced neurodevelopmental impairment of infants, the correlation analysis for cord blood lead level, the concentrations of dopamine metabolite homovanillic acid (HVA) and serotonin metabolite 5-hydroxyindoleacetic acid (5-HIAA) in cord plasma, and the neurodevelopmental scales of infants were conducted on 244 9-month-old children. Both score of sociability subscale and 5-HIAA concentration were correlated with cord blood lead level. The sociability score was negatively correlated with the concentration of HVA, whereas both coordination score and global score were negatively correlated with the concentration of 5-HIAA. With partial correlation analysis, after taking into account HVA, the significant negative correlation between the sociability score and cord blood lead level which existed in the linear correlation analysis disappeared, and the score of global scale negatively correlated with lead level in cord blood. When taking into account 5-HIAA, the scores of all the neurodevelopmental subscales except language subscale were significantly negatively correlated with lead level in cord blood. The results indicated that low level lead exposure in utero could produce the neurotoxic effect on developing serotonergic neurotransmitter system, which may underline the neurodevelopmental performances of 9-month-old children. Monoaminergic activity of central nervous system was suggested to have more potential effect on neurodevelopmental performances than low level lead exposure in utero.

P-56 THE EFFECT OF BENOMYL ON NEURITE OUTGROWTH FROM MOUSE AND HUMAN NEUROBLASTOMA CELLS IN VITRO McLean, W.G., Holme, A.D., Southgate, A.*, Howard, C.V.*, Reed, M.G.* Dept. of Pharmacology and Therapeutics, *Dept. of Pathology, University of Liverpool, Liverpool, U.K.

Benomyl (methyl 1-[(butylamino)carbonyl]-1H-benzimidazol-2-yl carbamate) is a commercially available fungicide. It acts by disrupting the function of microtubules. A link has been suggested between its consequent effects on neuronal morphology and its known teratogenicity. The purpose of this work was to determine the IC50 and the lowest observable effect levels (LOEL) of benomyl against a cytoskeleton-dependent function of both animal and human cultured neuronal cells, viz. differentiation-induced neurite outgrowth. Mouse NB2a and human SH-SYSY cells were grown in culture. Cells were plated on to 48- or 96-well plates at 16-20000 cells/ml; for SH-SYSY cells the wells were pre-coated with laminin. Cells were induced to differentiate by removal of serum and addition of 0.5mM dibutyryl cyclicAMP, with or without benomyl at concentrations from 0.001 to 10 mM. After 24h, cells were fixed and stained with Coomassie Blue and neurite outgrowth was quantified by image analysis. Two independent commercial preparations of benomyl significantly reduced neurite length with IC50 values (95% confidence limits) of 3.9 (1.4 - 10.6) x 10-6 M for NB2a cells and 1.0 (0.5-1.9) x l0-6 M for SH-SYSY cells. The LOEL was between 0.001 and 0.01 mM. We conclude that benomyl has potential neurotoxicity against developing neurones at low concentrations.

P-57 HEMATOLOGICAL AND NEUROPATHOLOGICAL ALTERATIONS IN DEVELOPING RATS DURING SINGLE AND COMBINED EXPOSURE TO CARBON MONOXIDE AND NITRATE Mavros, A., Stoltenburg-Didinger, G*, Lilienthal, H., Bilzer, T.**, Winneke, G. Medical Inst. of Environmental Hygiene at the Heinrich-Heine-University of Düsseldorf, *Inst. of Neuropathology at the University Hospital of Steglitz in Berlin, **Inst. of Neuropathology at the Heinrich-Heine-University of Düsseldorf, Germany

The aim of this study was the investigation of the effects by single and combined exposure to NO3 (300 mg/l drinking water) and CO (150 ppm) in developing rats starting at mating of the dams and ending on postnatal day (PND) 30. Pups were examined for carboxyhemoglobin (CO-Hb), methemoglobin (Met-Hb), total hemoglobin (t-Hb) levels and the morphology of neurons in the cortex and hippocampus (CA3) at PND1, PND10 and PND30. In the CO-exposed dams CO-Hb levels were 18.2% (controls: 1.55%), whereas in exposed pups it was 24.4% (controls: 2.7%) on PND1 and 22.6% (controls: 2.8%) on PND10. The t-Hb levels were significantly increased in the dams (17.2g/dl - controls: 15.1g/dl) and offspring on PND30 (14.2g/dl - controls: 12.1g/dl). Application of NO3 did not increase Met-Hb and did not alter t-Hb levels. Light microscopy of the brain sections of the CO and CO/NO3 groups on PND1 showed in general an increased size of neurons due to a small and distinct vacuolization both, intranuclear as well as within the neuropil. These findings were particularly expressed in the CA3 and cortical area. Such alterations were no longer apparent on PND10 and 30. Animals exposed only to NO3 did not differ from the controls. The additional exposure to NO3 did not result in an aggravation of the observed effects in hematological and histological examination. Preliminary findings of electron microscopy revealed cytoplasmic invaginations into the neuronal nuclei of the CA3 in newborn animals exposed to CO/NO3, resulting in an increase of nuclear surface. In the perikaryon the endoplasmic reticulum appeared less developed with less densely packed cisternae and ribosomes, indicating retardation of cell maturation.

P-58 TOXICITY OF THE STYRENE METABOLITE, PHENYLGLYOXYLIC ACID, IN RATS AFTER THREE MONTHS' ORAL DOSING Ladefoged, O., Lam, H.R., Ostergaard, G., Hansen, E.V., Hass, U., Lund, S.P.*, Simonsen, L.* Institute of Toxicology, *National Food Agency, National Institute of Occupational Health, Soborg, Denmark

Male Wistar rats were dosed with 0, 1250, 3750 or 5000 mg/L (w/v) phenylglyoxylic acid (PGA) in drinking water for 3 months (40 animal/group). There were no gross signs of toxicity or changes in clinical biochemistry that could be related to the test chemical. No changes in neurobehaviour were found in functional observational battery (FOB) or radial arm maze. The study revealed no changes in auditory brain stem response but minor changes in electroretinography. An effect on the relative weight of the kidney in the highest dose group was seen (0.504 ± 0.031 in the control group versus 0.579 ± 0.033 in the highest dose group). The histopathological studies revealed no changes in the structure of the kidney at the time of sacrifice. In the highest dose group, three animals showed slight changes in myelin sheet thickness in osmium stained tissue from peripheral nerves. No such changes were seen in the control group. A dose-dependent increase of dopamine (DA) concentration was found in hippocampus, hypothalamus, pons and medulla oblongata. The most marked changes were seen in hippocampus (0.56 ± 0.1 in the control group, 1.04 ± 0.11 in highest dose group, pmol/g tissue). A decrease in noradrenaline (NA) content was found in pons, from 4.12 ± 0.33 in control group to 3.62 ± 0.28 in highest dosed group (pmol/g tissue). The yield of synaptosomal protein and neurotransmitter concentrations were not affected. The males were mated with undosed females. No differences between groups were found in number of pregnancies, corpora lutea, implantations, live or dead fetuses or the frequencies of preimplantation loss. The role of PGA in the neurotoxicity of styrene is discussed.

P-59 GANGLIONEUROMAS OF RAT ADRENAL GLANDS Nedopitanskaya, N. Institute of Ecohygiene and Toxicology, Ministry of Health, Kiev, Ukraine

The medulla of adrenal gland is ectodermal in origin, being derived from the anlage of the sympathetic ganglia. Therefore, tumours of the medulla are neurogenic origin. According to literature data, phaeochromocytomas constitute most of the tumours of the adrenal medulla and ganglioneuromas are extremely rare compared to other adrenal tumours of rats. Own experience testify to more frequently of ganglioneuromas as "an extremely rare" in non-bread albino rats. They occur in rats over two years of age and are more frequent in males than in females. This report proposed the results of studies on a total of 680 rats (the group contained equal numbers of males and females). There are intact controls of routine chronic experiments for tests carcinogenic properties of chemicals in difference years. Ganglioneuromas have a very low incidence (4 from 680, it is 0,58 %) but phaeochromocytoma occurs in 1 incidence only (1 from 680, it is 0,15 %) and cortical adenomas are found in 5 animals from 680 (0, 74 %). It cannot be excluded that some tumours were lost, because only increased size adrenal glands were studied morphologically.

P-60 SEMAX (ACTH 4-10) INFUSION HASN'T AN INFLUENCE ON THE LEARNING OF RATS SUBJECTED TO HAEMORRHAGIC SHOCK Bastrikova, N.A., Novoderzhkina, I.S., Sokolova, N.A, Kamensky, A.A., Kozura, V.L. Institute of General Reanimatology, Moscow State University, Moscow, Russia

It was shown that haemorrhagic shock causes a number of changes in a week after arterial hypotension in rat's learning. Semax infusion doesn't influence these disturbances. Anesthetized (nembutal 40 mg/kg) rats were rapidly bled via a cateter in tail artery to a mean arterial pressure of 40 mmHg. Blood refletting or reinfusion was then instituted to maintain this pressure for one hour, followed by reinfusion with all shed blood. Then semax (7,5 mg/kg/min, group 3) or saline solution (0,8 ml in control, group 2) were infused intravenously via a cateter in jugular vein for 20 minutes. Rats were learned in T-like maze in a week. Four parameters were studied: the latent period of a reaction, the time of the right reaction, the number of the fulfilled reactions and the number of mistakes (the number of callings at the wrong compartment of the maze). Rats were learning four days. Reproduction was carried out in a week. In control group memory became worse in comparison with the group of rats (group 1) which were subjected to the same operation but were not bleeding. So in group 2 the number of mistakes under the reproduction increased sufficiently (P<0,05, Mann-Whitney-Wilcoxon test) comparing to group 1. The group 3 didn't differ from the group 2. Intravenous infusion of semax immediately after haemorrhagic shock didn't improve memory in a week.

P-61 THE INFLUENCE OF STRUCTURAL ANALOGUE OF ACTH4-10 HEPTAPEPTIDE SEMAX ON ACUTE HYPOBARIC HYPOXIA RESISTANCE IN ADULT AND NEWBORN WHITE RATS Balan, Polina V., Morozova, M.V., Krushinskaya, Ya.V., Sokolova, N.A. Dept. of Human and Animal Physiology, Faculty of Biology, Moscow State University, Moscow, Russia

It has been shown earlier that acute hypobaric hypoxia resistance in adult rats can be influenced by the structural analogue of ACTH4-l0 - heptapeptide Semax. In the present study we investigated the effect of this heptapeptide on hypoxia-induced changes in viability of newborn rats. Methods: Experiments were carried out on white adult and 7 days old rats. An acute hypobaric hypoxia was induced in barocamera on 11 500 m of altitude. The main parameters of evaluation were: life time (was considered to be before breath stop), restitution time (was registered just after breath stop until pose restoration) and life time/restitution ratio. Semax and equivalent volumes of physiological saline were injected 15 min before hypoxia. Control restitution time had a mean level of 221.3 sec in adults and 44 sec in newborn. Thus, time of restoration after an acute shock is significantly less in newborn rats than in adults supporting the idea of higher resistance of pups to acute hypoxia as compared to adults. 0.1 mg/kg injection of the heptapeptide Semax caused 1.8 fold increase in life time whereas 0.05 mg/kg had no effect on registered parameters in adult rats. At the same time 0.05 mg/kg dose caused significant (p<0.01 ) increase in life time and life time/restitution ratio in newborn rats. In conclusion, dose of 0.5 mg/kg which appears to be subthreshold in adults causes significant improvement of acute hypobaric hypoxia resistance in 7days old rats.

P-62 THE DELAYED BEHAVIORAL CONSEQUENCES OF 12 MIN CARDIAC ARREST-TYPE CEREBRAL ISCHEMIA IN RATS Nazarenko, Irina V., Kamensky, A.A., Volkov, A.V. Institute of General Reanimatology, Moscow, Russia

It is widely known that sudden total circulatory arrest and following reperfusion lead to severe damage of the central nervous system and to development of postischemic-anoxic encephalopathy. On the clinical data psychoneurological complications are observed in more than 70 % of cases in early and delayed stages of postischemic period. In the present study we investigated the behavioral performances of rats after global cerebral ischemia induced by 12-min cardiac arrest according to the method originally designed by Korpachev et al.(1982). Two weeks after cardiac arrest the behavioural status was investigated using Rodina's et al. (1993) method for estimation of the anxiety-phobic states in rats, open field, T-maze, tail-flick and hot-plate. The mortality of rats after cardiac arrest was 30 %. Global cerebral ischemia did not cause significant change in the linear locomotion, whereas rearing and number of open field crossings were markedly increased compared with intact animals, while defecation number was decreased. As for emotional characteristics the reduction of anxiety-phobic index has been found. In T-maze cardiac arrest increased number of correct trials and decreased latency, but failed to change time of reaction. The pain sensitivity of ischemic rats in tail flick did not differ from intact ones and was significantly reduced in hot plate. The data obtained suggest that cardiac arrest-type cerebral ischemia resulted in the disturbance of rats' emotional reactivity and their estimation of environmental stimuli significance.

P-63 THE DOSE-RESPONSE RELATIONSHIPS IN NEUROPSYCHOLOGICAL STUDY OF CS2 EXPOSED WORKERS Bazylewicz-Walczak, Barbara The Nofer Institute of Occupational Medicine, Lódz, Poland

Cross-sectional study was carried out on 310 men -155 exposed to CS2 from 4 to 36 years, employed in viscose fibre factory, and 155 unexposed blue-collar workers matched with respect to age and education. The subjects were examined with the use of WHO Neurobehavioral Core Test Battery. Life dose of exposure to CS2 was calculated for every worker. It ranged from 41 to 742 (year · mg/m3). Multiple regression analysis, made for all subjects (N = 310) revealed that independent variable "exposure" explains variation of 31 psychological variables from among 32 ones controlled in the study. Workers exposed to CS2 with the lowest range of life-dose (41-200) did achieved significantly worse test results in 29 from 32 psychological variables than controls.

P-64 HUMAN NEUROTOXICITY RESEARCH METHODS: BEHAVIORAL TESTING IN DIFFERENT CULTURAL GROUPS Anger, W.K., Sizemore, O.J., Grossmann, Sandra J., Rohlman, Diane S., Glasser, Julie A. Center for Research on Occupational and Environmental Toxicology, Oregon Health Sciences University, Portland, OR, USA

Behavioral tests from the two consensus neurotoxicity test batteries (established by the World Health Organization and the US Agency for Toxic Substances and Disease Registry) were administered to 715 people aged 26-45, with 0-18 years of education, from four cultural groups in the USA: European-descent majority, Native American Indian, African-American, and Latin-American populations. Performance on the behavioral tests used extensively in worksite and environmental research to detect neurotoxic effects, was heavily influenced by subject variables, particularly education and cultural group. Based on this series of studies, neurotoxicity tests selected for a cross-sectional assessment of Latin American workers exposed to pesticides were administered in a series of pilot studies. This led to extensive modifications of test protocols, changes in initial Spanish language translations (the source tests were developed and validated in English-speaking European populations), and substitution of tests to maximize the effectiveness of the assessment. The evaluative process and the outcomes will be described.

P-65 ASSOCIATION OF BIOLOGICAL AND SUBJECTIVE INDICATORS OF DRINKING HABITS WITH THE PERFORMANCE IN DIFFERENT NEUROBEHAVIORAL TASKS van Thriel, Ch., Zupanic, M., Demes, P., Willer, H., Seeber, A. Institute of Occupational Physiology at the University of Dortmund, Germany

There is some empirical evidence that mnemonic deficits, especially on recent memory functions are associated with damages of the hippocampal system. Exposure to occupationally used neurotoxic agents e.g. organic solvents, heavy metals, and alcohol abuse were considered to be sources of damages in the hippocampus. The present study is part of a longitudinal study among male rotogravure printers occupationally exposed to toluene and control subjects who were tested in four computer-based neurobehavioral tasks. Habitual alcohol consumption as a confounder of exposure effects was assessed by (a) biological marker, i.e. carbohydrate- deficient-transferrin (CDT) and gamma-glutamyltransferase (GGT) and (b) subjective marker, i.e. reported drinking habits in g/day. As a first result a positive correlation between both types of marker is observable. Secondly, while occupational exposure to toluene had no effect on any neurobehavioral performance test, biological markers of improved habitual drinking did have. The combined utilization of values in CDT and GGT as biological indicators of increased drinking habits leaded into an matched group comparison within a small subgroup of all participants. Matching procedure included other confounders, i.e. age and verbal intelligence. Individual results of neurobehavioral tasks with enhanced memory involvement were impaired in the group with enhanced CDT and GGT values, while simple and choice reaction time tasks were not effected. From this results it could be hypothesized that habitual higher alcohol consumption may impair memory function more frequently than other CNS functions. Surprisingly, there was a positive relationship between subjective and biological marker of habitually increased alcohol consumption, but the results also stressed the significance of these objective markers of in neurobehavioral research.

P-66 DISCRIMINANT VALIDITY OF A NEW QUESTIONNAIRE FOR CHEMICAL AND GENERAL ENVIRONMENTAL SENSITIVITY Kiesswetter, E., Sietmann, B., Golka, K., Zupanic, M., Seeber, A. Institute of Occupational Physiology, University of Dortmund, Germany

Individual physical and psychological characteristics are under discussion to explain severe reactions on low level exposure to chemical agents. A questionnaire approach was used to study some relevant aspects of sensitivity. Sensitivity was defined as strong reactions on physical environmental influences (e.g. noise, heat), chemical odours (e.g. paints, car exhaust), and natural substances (e.g. pollen, food). Chemical exposed and non exposed workers completed the new sensitivity questionnaire and two further questionnaires on neurotoxic symptoms and anxiety (n = 600). According to the construction of the sensitivity questionnaire, three main dimensions could be extracted by factor analysis which were labelled general environmental sensitivity (GS), multiple chemical sensitivity (MCS), and allergy (A). The prevalence of self-reported chemical sensitivity and allergy amounted to 19 % and 28 % of the study population, respectively. The discriminant validity of the questionnaire was examined referred to: (1) groups with different chemical exposures, (2) odour thresholds, (3) neurotoxic and anxiety symptoms. Different chemical exposures (e.g. single solvents, solvent mixtures, dioxins) showed nearly no influence on the prevalence of chemical sensitivity and allergy. The correlation between MCS and odour perception was only small. However, insensitive and sensitive individuals could be differentiated by characteristic symptom complexes corresponding to a biological-psychological model. Allergy was related to irritative, MCS and GS to psychological-neurological and anxiety symptoms.

P-67 NEUROBEHAVIORAL PERFORMANCE OF CZECH SCHOOL CHILDREN BORN IN YEARS OF MAXIMAL AIR POLLUTION (1982-1983) Otto, D., Skalik, I.*, Bahbouh, R.**, Hudnell, K., Sram, R.* National Health and Environmental Effects Research Lab., USEPA, RTP, USA, *Regional Inst. of Hygiene for Central Bohemia, **Dept. of Psychology, Charles University, Prague, Czech Republic

Ambient levels of SO2, NOx, PAHs and heavy metals are elevated in Northern Bohemia as a result of intensive mining and combustion of brown coal. SO2 levels, a general measure of air pollution, were highest in 1982 and 1983. Sram (1991) hypothecized that in utero exposure to chemicals causes functional changes in the nervous system expressed as developmental disorders or behavioral impairments. To test this hypothesis, tests from the Neurobehavioral Evaluation System (NES2) were administered to 519 7th-grade children from three districts with varying levels of air pollution. Questionnaires were administered to parents and teachers to assess SES, neonatal health history and school performance. Hair and urine samples were also obtained from children to measure arsenic and mercury exposure. Children from Teplice, the heavily polluted mining district, performed more poorly and were referred more often for assessment of learning disorders than children from Prachatice--an agricultural district--or Znojmo--a district where natural gas is used for heating and power generation. Digit span and symbol-digit scores also varied significantly with hair As and Hg levels DISCLAIMER: This is an abstract of a proposed presentation and does not necessarily reflect EPA policy.

P-68 INCREASED MEDICATION USE IN A COMMUNITY ENVIRONMENTALLY EXPOSED TO CHEMICALS Bowler, R.M., Hartney, C., Ngo, Long*, Rauch, S San Francisco State University, *University of California Berkeley, El Cerrito, USA

An epidemiological health study compared health status in an exposed and control town each with about 3300 residents, six months after a 16 day Catacarb chemical release from an adjacent oil refinery. Constituent components of Catacarb are metavanadate, diethanolamine, potassium carbonate and boron. Few human studies exist, however animal studies have shown these chemicals to be respiratory, gastro-intestinal, dermatologic and visual irritants. Overall household response rate was 43%. After controlling for effects of gender, education, race and household cluster, increases (p<0.05) in respiratory, visual, and dermatological symptoms were found (O.R. 1.3-3.0). Illnesses were reported to be worse after the release in the exposed town for acute and chronic bronchitis (Risk difference 6.7%, 14.3%); asthma (O.R. 4.6); hayfever (O.R. 2.4); sinus trouble (O.R. 3.0); allergies (O.R. 4.7), skin rashes (O.R. 2.0); eye (O.R. 4.3); bladder infection (O.R. 5.6). Although medication use was similar in both towns prior to the release, significantly increased medication use after the release was reported for antacid, and asthma, cough and cold, eye, headaches and sleep medicaiton for the exposed town. Increased dose of medication resulted in adjusted prevalence ratios of 2.5 for antacid, 2.7 for asthma, 2.0 and 2.1 for cold and cough, 3.7 for headaches, 2.7 for eye, 2.9 for skin and 4.8 for sleep. In conculsion, this community is shown to suffer negative health consequences of the chemical exposure in increases in symptoms, worsening of illnesses and increased medication use.

P-69 PREDICTIVE VALIDITY OF THE Q16 QUESTIONNAIRE: A COMPARISON BETWEEN REPORTED SYMPTOMS AND NEUROBEHAVIORAL TESTS Bergamaschi, E., Smargiassi, A., Mutti, A., Cella, M.T., Franchini, I. Department of Clinical Medicine, Nephrology and Health Sciences, University of Parma, Italy

The correspondence between the subjective answer to the Q16 questions relevant to memory and attention-concentration and the objective performance on neurobehavioral testing has been evaluated; moreover, the sensitivity of Q16, its specificity and predictive validity, both positive and negative, have been assessed taking the neurobehavioral test score as a reference diagnostic criterion, the lower quartile of neurobehavioral performance being considered as a positive (abnormal) response. The group under study consisted of 74 volunteers (24 females), recruited among workers exposed to styrene and among healthy controls, aged 40 years on average (SD: 7.5). The test battery included the logical memory (short- and long-term) and the verbal learning (short- and long-term) tests of the Wechsler Adult Intelligence Scale (WAIS). (i)The answers to the Q16 questions were only partially related to the performance of neurobehavioral tests: self-perceived forgetfulness showed a limited agreement with the long-term logical memory test (p<0.05). (ii) The number of false negatives (no symptoms but low test scores) was generally high for relevant answers to Q16 questions, giving rise to a very low sensitivity of the questionnaire, despite a relatively high specificity. Accordingly, the positive diagnostic validity was low (<30%), whereas the negative diagnostic validity was high (>80%). Therefore, a positive answer to the Q16 questions does not predict a poor performance in neurobehavioral tests. The lack of a good correspondence between different methods to investigate subtle neurological changes may reflect an inconsistency between self-perceived disturbances and objective measurements of relevant functions. In order to detect early adverse effects, a positive answer to the Q16 questions is less sensitive than neurobehavioral tests. Aknowledgments: Supported by the European Union (Contr. No. ENV4-CT96-0173)

P-70 OCCUPATIONAL AND ENVIRONMENTAL RISK FACTORS FOR PARKINSON'S DISEASE IN THE EMILIA-ROMAGNA REGION OF NORTHERN ITALY Smargiassi, A., Mutti, A., De Rosa, A., Negrotti, A.*, Calzetti, S.* Laboratory of Industrial Toxicology, Department of Clinical Medicine, Nephrology and Health Sciences, *Inst. of Neurology, University of Parma Medical School, Parma, Italy

To investigate possible risk factors for Parkinson's disease (PD) in the Emilia-Romagna region, a questionnaire-based case-control study was carried out on 50 patients with neurologist-confirmed idiopathic PD and 50 controls similar for sex and age. To avoid a potential selection bias, the control group was recruited in specialistic centers of the same University Hospital (glaucoma, psoriasis vulgaris and essential arterial hypertension). A positive exposure was defined as occupational or residential contact with a given factor for at least 10 consecutive years prior to the onset of PD. Smoking habits was defined by exclusion of those subjects who never smoked. The following risk factors were identified: rural residency (OR: 9.33; 95% CI: 1.99-43.68), well-water drinking (OR: 4.47; 95% CI: 1.75-11.43), exposure to copper sulfate, largely used in the Italian viticulture (OR: 3.60; 95% CI: 1.44-8.91) and jobs implying exposure to metals, organic solvents or pesticides (OR: 3.86; 95% CI: 1.67-8.91). Smoking habits was negatively associated with PD (OR: 0.24; 95% CI: 0.10-0.55), confirming many studies indicating a "protective" role of tobacco smoking. However, though a spontaneous selection is unlikely, the prevalence of subjects who never smoked was much lower in the control group, as compared to the general population. As a whole, these results support the role of environmental factors in the etiology of PD. Owing to the limited sample-size, a detailed analysis of individual risk factors and their interactions cannot be performed. A larger study including several genetically determined potential risk factors is in progress. Acknowledgments: Supported by the European Union (ENV4-CT-96-0173), and by a Québec IRSST scholarship.

P-71 THE INFLUENCE OF DRINKING CUSTOMS TO THE HEALTH STATE OF ALCOHOL-ADDICTED PEOPLE Gömbös, Teodóra, Móritz, P. Medical Experts' Institute of the Hungarian State Railways, Budapest, Hungary

The health state deterioration of alcohol-addicted people depends not only from the consumed quantity of the aethyl alcohol, but also from the quality of the consumed alcoholic drinks. The Hungarian alcohol-addicted people, above a1l the perunious layer is endangered by the wines, fermented from Labrusca-type grape and by brandies originating from the illegal distilleries. The methyl alcohol in the Labrusca-type wines and the toxic agents, remaining in the single distilled brandies are the main factors causing rapid developlment of hepatic cirrhosis and encephalopathy in this population. 5 years of such kind of alcohol-abuse led to a total deterioration and disability to work. Such a rapid and severe damage of the health state is not seen by people drinking quality wine or brandies.

P-72 EXPOSURE TO 60HZ ELECTROMAGNETIC FIELDS INCREASES THE PROLIFERATION OF HUMAN ASTROCYTOMA CELLS Costa, L.G., Wei, M., Guizzetti, M. Department of Environmental Health, University of Washington, Seattle, WA, USA

Epidemiological studies have provided evidence that exposure to electromagnetic fields (EMF) may be associated with an increased incidence of brain tumors, most notably astrocytomas. In this study we investigated whether exposure of human 1321N1 astrocytoma cells to 60HZ EMF would affect their proliferation. Proliferation was measured by 3H-thymidine incorporation, flow cytometry and cell counting. Exposure to EMF was carried out in a double-sided incubator, using a one-coil Meritt system. EMF (1.2 Gs for 24 or 48h) increased proliferation of astrocytoma cells by 80% and strongly potentiated the effect of two mitogens, the muscarinic agonist carbachol and the phorbol ester TPA. Bisindolylmaleinimide, of PKC inhibitor, inhibited the effect of EMF on basal proliferation and completely antagonized it's potentiation of carbachol- and TPA-stimulated proliferation. These results indicate that EMF exposure can increase the proliferation of human astrocytoma cells and potentiate the effect of mitogens. These findings may offer a biological basis for the observed epidemiological association between EMF exposure and brain tumors. Supported in part by ES-07033 and ES-08851.

P-73 EFFECTS OF PRE- AND POSTNATAL PCB-EXPOSURE ON PRESPEECH DEVELOPMENT IN SEVEN MONTH OLD INFANTS* Wiener, J.A., Bisping, R., Heinzow, B.*, Krämer, U.**, Steingrüber, H.-J., Winneke, G.** Institute of Medical Psychology, University of Düsseldorf, *State Agency of Nature and Environment Schleswig-Holstein, Flintbeck by Kiel, **Medical Institute for Environmental Hygiene at the University of Düsseldorf, Germany

PCBs are considered as potential hazard for the growing infant, but findings on neuro- or cognitive development are inconsistent. To supplement, the possible effects of PCB-exposure on prespeech development were explored. Data. were collected in 163 seven months olds from the Düsseldorf area. PCB-concentrations (S 138, 153 and 180) were measured in cordblood and maternal milk. Vocalizations were video recorded for 10 minutes. Based on nonvegetative comfort utterances only, the total time of vocalization (i.e. Articulation Activity: AA) and the Articulation Level (AL) were determined. AL was judged by a 5-point Behaviorally Anchored Rating Scale with category representing the lowest and category 5 the highest level. Prototypes were checked to fulfil the requirements of the corresponding category by high resolution FFT-spectrography, band-limited analysis, and listening. AL-scoring was based on comparison with prototypes. Interrater reliability was r = .98 for AA and r³.95 for AL. PCBs in milk and AL were negatively associated (r = -.l7; p = .06 two tailed). The result suggest, that - using PCB-exposure as an environmental model - the level of articulation as a highly reliable measure might be a sensitive indicator for a subclinical delay of development. This work was part of a study supported by the EU (Brussels), Contract: EV5V-CT 92-0207.

P-74 NEUROBEHAVIORAL DEVELOPMENT IN HUMAN INFANTS IN ASSOCIATION WITH PCBs IN THE NEONATAL PERIOD Walkowiak, J. , Bucholski, A., Heinzow, B.*, Krämer, U., Plaßmann, S., Schmidt, E.**, Steingrüber, H.-J.***, Wiener, A.***, Winneke, G. Med. Inst. of Environmental Hygiene, Heinrich-Heine-University of Düsseldorf (HHU),*State Agency of Nature and Environment, Schleswig-Holstein, Flintbeck by Kiel, **Medical School, Children's Hospital, HHU ***Inst. of Med. Phychology, HHU, Düsseldorf, Germany

Some polychlorinated biphenyls (PCBs) or their metabolites are known to cross the placenta. After birth the infant can be exposed through breastfeeding. The effects of pre-and postnatal PCB-exposure on neurobehavioral development are still uncertain (Seegal, 1996). Methods: A European coordinated study was initiated including cohorts from Denmark (Faroe Islands), Germany (Düsseldorf) and the Netherlands (Groningen, Rotterdam). PCB-congeners 138, 153, and 180 were measured in cordserum and maternal milk, and neurobehavioral examinations were done at 2 weeks, 7 and 18 months. Results from the Düsseldorf cohort only will be presented. 171 healthy mother-infant pairs were recruited. The median PCB-concentration (S 138, 153, 180) in cordserum was 530 ng/l (range: 170 - 1360 ng/l), and in maternal milk at two weeks 405 ng/g fat (range: 64 - 944 ng/g). After control for relevant confounders no significant associations were found between PCBs in cordserum and any of the target variables (anthropometry, neurological status according to TOUWEN/ PRECHTL, mental or motor development according to the BSID II, or visual recognition memory according to FAGAN). Only for maternal milk a negative borderline association was found between PCBs and mental development (BSID II) at 7 months of age. It is concluded that the results to date of the Düsseldorf cohort are partly at variance with some previous reports about the neurobehavioral toxicity of PCBs at background exposure levels. Supported by the EU (Brussels), contract: EV 5 V- CT 920 207

P-75 BEHAVIORAL CONSEQUENCES OF IN UTERO EXPOSURE TO SINGLE AND COMBINED PCB-CONGENERS IN RATS Hany, J., Lilienthal, H., Weinand-Härer, A., Winneke, G. Medical Institute of Environmental Hygiene, Department of Biological Physiology, Düsseldorf, Germany

The aim of the present experiments was to compare behavioral and neurochemical effects resulting from prenatal administration of two single PCB-congeners and their combination. Pregnant Long Evans rats received daily injections with the coplanar 3,4,3',4'-TetraCB (PCB77; 1.5 mg/kg or 0.5 mg/kg), the ortho-chlorinated 2,4, 2',4'-TCB (PCB47; 1.5 mg/kg) or a combination of both congeners (0.5 mg PCB77 + 1.0 mg PCB47 /kg) from day 7-18 of gestation. Behavioral tests were carried out in different life stages in the offspring. In an open field experiment the preference for the inner zone seemed to be affected on postnatal day (PND) 70 in rats prenatally exposed to 1.5 mg/kg PCB77. In the 0.5 mg/kg PCB77 group a less pronounced but similar effect could be observed on PND18. After one year the activity of control animals was clearly reduced in comparison to all PCB-treated groups. In a passive avoidance task shorter latencies could be observed 5 min after the acquisition in the groups exposed to 1.5 mg/kg PCB77 or to the combination of both congeners. When tested for haloperidol-induced catalepsy PCB77-rats showed longer latencies to movement onset 60 min after injection of the dopamine receptor blocker haloperidol. In conclusion, most of the observed long-lasting effects appeared to be dependent on perinatal PCB77 exposure while PCB47 was less effective. Combined exposure did not alter or even diminished the observed effects. Supported in part by the Dept. for Environment, Baden-Württemberg, FRG, grant No. PUG U 95 003 to H.L.

P-76 EFFECTS OF POLYCHLORINATED BIPHENYLS (PCB) ON THE NERVOUS SYSTEM Voie, O., Andersen, J., Farnes, C., Fonnum, F. Norwegian Defence Research Establishment, Division for Environmental Toxicology, Kjeller, Norway

Ortho substituted PCB congeners have toxic effects on the nervous system. Low concentrations of 2,2'-DCB stimulated glutamate release from synaptosomes and inhibited the uptake of the neurotransmitter dopamine in the synaptic vesicles. 2,2'-DCB elevated [Ca2+]i in synaptosomes. We suspected some of these effects to be due to interference with the intracellular signal systems. The effect of PCB on the Ca2+-homeostasis and production of free radicals in human granulocytes was investigated as a model for the nervous system. Intracellular free calcium, [Ca2+]i, was increased in a dose-dependent manner by 2,2'-DCB. The increase in [Ca2+]i in response to 2,2'-DCB was Ca2+-dependent and phospholipase C-dependent, an enzyme of the inositol phosphate transduction pathway. In human granulocytes Ca2+-influx occurs by activation of Ca2+ release activated Ca2+ channels (CRAC) in the plasma membrane. We suggest that PCBs stimulate such a Ca2+ release from intracellular stores by production of Ins(1,4,5)P3 and/or by inhibition of Ca2+-ATPase. Respiratory burst, measured as chemoluminescence was stimulated in a dose-dependent by 2,2'-DCB In absence of extracellular calcium, the respiratory burst in response to 2,2'-DCB and 2,2',4,4'-TeCB was reduced by 63% and 82%. Neomycin, which inhibits phospholipase C had no significant effect on the respiratory burst in response to PCB. Ethanol (1 %), a phospholipase D modulator reduced the effect on the respiratory burst in response to 2,2'-DCB and 2,2',4,4'-TeCB by 72% and 75%, respectively. Wortmannin (25 nM), a tyrosine kinase inhibitor and an inhibitor of activation of phospholipase D erased the effect on the respiratory burst in response to PCB. We conclude that PCBs activate respiratory burst through a transduction pathway that involves a tyrosine kinase activation, possibly downstream of the chemoattractant receptor, a phospholipase D activation and a protein kinase C activation prior to activation of the NADPH oxidase.

P-77 SINGLE OR COMBINED MATERNAL EXPOSURE TO A COPLANAR AND AN ORTHO-CHLORINATED PCB CONGENER IN RATS: IMPAIRMENTS OF LEARNING IN THE ADULT MALE OFFSPRING Weinand-Härer, A., Lilienthal, H., Hany, J., Winneke, G. Medical Institute of Environmental Hygiene, Heinrich-Heine-University, Düsseldorf, Germany

It is reported that prenatal exposure to PCBs causes neurobehavioral deficits in animals and humans. The reasons for these changes may be alterations of thyroid hormone levels and/or changes of the dopaminergic system during neuronal development because both systems are influenced by PCBs. Time-mated rats were subcutaneously injected daily with the coplanar 3,4,3',4'-TCB (0.5 mg/kg b.w.), the ortho-chlorinated 2,4,2',4'-TCB (1.5 mg/kg b.w.), with the combination of both PCBs, or with the vehicle (olive oil) from gestational day 7 to 18. Male rat offspring were tested in a radial arm maze for spatial learning at the age of ten months. On the second day of training, time to find all pellets was significantly reduced in the group exposed to the coplanar PCB congener in comparison to controls. The rats of the coplanar group also preferred directly adjacent arms. Accuracy was not effected by either treatment. Rats of the coplanar group and the combination group had a significant higher locomotor activity than control rats in the open field. Thus, the differences during maze learning may be due to enhanced locomotor activity. At about 12 months of age naive male offspring were tested for conditioned taste aversion using a solution of 0.1% saccharin as the conditioned stimulus and estradiol (10 µg/kg b.w.) as the unconditioned stimulus. Saccharin aversion was significantly less pronounced in the coplanar and the combination group in comparison to the controls. The results suggest that the amygdala rather than the hippocampus may be affected by maternal exposure to PCBs. Supported in part by the Dept. for Environment, Baden-Württemberg, FRG, grant No. PUG U 95 003 to H.L.

P-78 EFFECTS OF MATERNAL EXPOSURE TO SINGLE AND COMBINED PCB CONGENERS ON THE ELECTRORETINOGRAM IN RATS Lilienthal, H., Kremer, H., Weinand-Härer, A., Hany, J., Winneke, G. Medical Institute of Environmental Hygiene, Department of Biological Physiology, Düsseldorf, Germany

The purpose of this pilot study was to examine PCB-induced effects on the visual system. Pregnant Long Evans rats were injected daily with 2,2',4,4'-tetrachlorobiphenyl (PCB-47; 1.5 mg/kg), 3,3',4,4'-tetrachlorobiphenyl (PCB-77; 1.5 mg/kg), or a combination of both congeners (1.0 mg PCB-47+0.5 mg PCB-77/kg) from gestational day 7 to 18. Measurements of the electroretinogram (ERG) started in the offspring at an age of about 200 days. After a dark adaptation period of 40 min rats were sedated with ketamine and xylazine. At first four scotopic potentials were recorded after flash stimulation with increasing luminances at Ganzfeld conditions. Then the maximum potential and oscillatory potentials were measured, followed by flicker responses and the cone response after turning on the background lights. The amplitude of the b-wave was diminished in scotopic potentials and in the maximum potential in females exposed to PCB-77. In addition, the amplitudes of the first two wavelets of the oscillatory potentials and of the first flicker response taken immed-iately after the end of the dark period were decreased in these animals. In contrast, no differences between groups were found in males. The results suggest an alteration of the ERG by PCB-77 in female rats which cannot be ascribed to the total dose of PCB. Supported in part by the Dept. for Environment, Baden-Württemberg, FRG, grant No. PUG U 95 003 to H.L.

P-79 ALTERATIONS IN MEMBRANE PROPERTIES OF CULTURED BOVINE CHROMAFFIN CELLS AFTER EXPOSURE TO THE ORTHOCHLORINATED 2,2';4,4'TCB Bickmeyer, U., Wiegand, H. Dept. of Neurotoxicology, Medical Institute of Environmental Hygiene, Düsseldorf, Germany

Experiments were performed at bovine chromaffin cells in short term culture. Electrophysiological experiments were done during the first three days in culture using the patch clamp technique in the whole cell configuration. In physiological buffer as external solution and standard internal pipette solution, we measured the membrane potential and membrane currents under defined conditions. Cells hyperpolarized after application of 100 M TCB by values of about -5 to -10mV. Cells voltage clamped to membrane potentials of -20mV showed an outward current induced by application of TCB. Elevation of the concentration of free calcium ions may trigger an outward current probably carried by potassium. If the measured effects are a result of specific actions of the congener or more unspecific actions of the substance in combination with the vehicle DMSO remains to be solved. We are in the progress to characterize these effects in detail.

P-80 TIME-DEPENDENT EFFECTS OF AN ORTHO-SUBSTITUTED PCB CONGENER ON CATECHOLAMINE RELEASE OF BOVINE CHROMAFFIN CELLS Messeri, M.D., Bickmeyer, U., Müller, E., Redelings K.-H., Wiegand, H. Medical Institute of Environmental Hygiene, Heinrich-Heine-University, Düsseldorf, Germany

We have previously compared the effects of 2 PCB-congeners with different substitution patterns on the catecholamine release and -content of primary cultures of bovine chromaffin cells. The ortho-substituted congener 2,4 TCB enhanced the spontaneous catecholamine release and reduced the total catecholamine content of the cells after 24 hours- and 5 days-exposure. On the contrary the coplanar congener had no effects on the catecholamine release and content of the cells. We have investigated the time-dependency of the spontaneous catecholamine release of chromaffin cells during exposure to 2,4 TCB 100 M. The spontaneous release was increased at the day 1 and 2 up to 430 % and was reduced from the 3rd to the 5th day. We have also investigated the time dependency of the spontaneous catecholamine release during 12 hours of exposure to 2,4 TCB 100 M. We observed an increase after 1 hour of exposure until the 4th hour, then the spontaneous release remained constant until the 12th hour. We measured the Ca2+-levels of individual cells during 10 minutes of exposure to 2,4 TCB 100 M. We observed an elevation of the Ca2+-levels in Ca2+-containing buffer.

P-81 CONGENER-SPECIFIC EFFECTS OF MATERNAL PCB EXPOSURE ON FUNCTIONAL PLASTICITY IN CORTICAL AND HIPPOCAMPAL SLICES OF RATS Altmann, L., Lilienthal, H.*, Hany, J.*, Wiegand, H. Dept. of Neurotoxicology, *Dept. of Psychophysiology, Medical Instiute of Environmental Hygiene, Heinrich-Heine-University, Düsseldorf, Germany

The developing nervous system is considered to be particularly affected by exposure to polychlorinated biphenyls (PCBs). We exposed rats prenatally to the coplanar congener 3 3 '4 4'-tetrachlorobiphenyl (3,4-TCB) or to the ortho-chlorinated congener 2 2' 4 4'-tetrachlorobiphenyl (2,4-TCB) and examined functional plasticity in the brains of the young as well as the adult offspring in vitro. Pregnant rats received daily injections of TCB dissolved in oil or the same amount of as vehicle (controls), respectively, at days 7 to 18 of gestation. The offspring of each exposure group was investigated electrophysiologically at postnatal days 11 to 19 (young) or as adults. The amount of long-term potentiation (LTP) as well as paired-pulse potentiation was determined in slices from the visual cortex and hippocampus of these animals. While in the cortical slices from the controls LTP was found following high-frequency stimulation (HFS) the 3,4-TCB exposed rats expressed long-term depression (LTD) instead of LTP. In 2,4-TCB exposed rats HFS resulted in either LTP, LTD or no change. In the hippocampal region, no statistically significant differences in the amount of LTP could be measured between the controls and the 2,4-TCB or 3,4-TCB exposed rats. Our results suggest that the visual cortex is functionally altered by the coplanar 3,4-TCB even at low exposure levels if the exposure takes place during embryonic development. The hippocampal region seems to be able to compensate for PCB induced effects.

P-82 COGNITIVE DEFICITS IN SOLVENT-EXPOSED WORKERS: CONTROLLING FOR VISUAL AND MOTORIC INFLUENCES ON COMPUTERIZED TEST RESULTS Hudnell, H.K., Otto, D.A., House, D.E., Darcey, D.J.*, Broadwell, D.O.*, Boyes, W.K. U.S. Environmental Protection Agency, *Duke University Medical Center, USA

Computerized tests of neurobehavioral functions are widely applied in environmental-epidemiological studies to evaluate the human-health risks of exposures to neurotoxicants. Many tests are designed to assess specialized cognitive functions. However, the results also depend on visual and motoric functions since stimuli are presented visually and motoric responses are required. Statistical analyses which do not take into account visual and motoric influences on test performance may lead to erroneous attributions of exposure-related changes to cognitive deficits. Sensory, motoric and cognitive functions were assessed in 25 microelectronics workers chronically exposed to organic solvents and 32 age, gender, education and ethnicity-matched control subjects. ANOVA procedures showed significant exposure-induced deficits in visual contrast sensitivity (VCS) and finger tapping (FT) speed. Regression analyses on data from control subjects described relationships between VCS, FT and the cognitive tests. The regression coefficients were used to adjust cognitive test scores of all subjects for differences in VCS and FT, thereby removing these influences on the results of cognitive test. The results indicated that group differences in the Simple Reaction Time and Hand-eye Coordination tests could be attributed to visual and motoric deficits in the exposed workers. However, visual and motoric functions were unable to explain group differences in five tests of cognitive functions. Solvent exposure in the microelectronics industry apparently induced persistent deficits in the cognitive functions of coding, attention, pattern memory, and verbal fluency. This abstract has been cleared for publication but does not necessarily reflect Agency views.

P-83 BIOMARKERS OF EXPOSURE TO SOLVENT MIXTURES: CO-EXPOSURE OF RATS TO N-HEXANE AND ACETONE M.dos Santos, Ana P., Mateus, M.L., C.Batoréu, M.C. Faculty of Pharmacy, Laboratory of Toxicology, Lisboa, Portugal

The toxic effects resulting from human exposure to solvent mixtures, have been insufficiently studied ; however this subject deserves special attention as workers are often exposed to mixtures of solvents which can interact producing a synergistic or antagonistic effect. Acetone potentiates the neurotoxicity of n-hexane in rodents after chronic exposure to n-hexane plus acetone. n-Hexane is metabolized to 2,5-hexanedione (2,5-HD) and this diketone reacts with amino groups of neurofilaments yielding pyrrolic adducts responsible for n-hexane neuropathy. In this study, the changes of excretion of n-hexane derivatives in urine of rats - free and conjugated 2,5-HD, and pyrroles - are used to investigate the interaction between n-hexane and acetone. Six groups of male Wistar rats were exposed daily by gavage to: n-hexane, acetone, and n-hexane plus acetone (four increasing doses of acetone). Free and conjugated 2,5-HD and pyrroles were determined in urine using GC/FID and Ehrlich's reagent respectively. After the first day of exposure all the groups exposed to the mixture of solvents showed no significant changes in the excretion of the three biomarkers analysed. After the second day of exposure there is a signiflcant increase in excretion of free 2,5-HD and pyrroles when compared with those derivatives excreted in urine of rats exposed only to n-hexane. After the third day of exposure there is also a significant increase of conjugated 2,5-HD in urine. The results obtained suggest that there is an earlier detection of neurotoxicíty potentiation resulting from the combined solvent exposure, when it was used free 2,5-HD and pyrroles as biomarkers of exposure to n-hexane.

P-84 DEFICITS ON A SPATIAL NAVIGATION TASK FOLLOWING PRENATAL EXPOSURE TO TOLUENE Hougaard, K.S., Hass, U., Lund, S.P., Simonsen, L. Department of Toxicology and Biology, National Institute of Occupational Health, Copenhagen, Denmark

The developmental and neurobehavioural effects of prenatal exposure to toluene (CAS 108-88-3) were studied after exposing pregnant rats (Mol:WIST) to 1800 ppm toluene for 6 hours daily on days 7-20 of gestation. Body weight gain was slightly, but nonsignificantly suppressed in treated dams during the period of exposure. Body weights of exposed offspring were lower until day 10 after parturition. Surface righting was retarded on days 2, 4 and 5, but this can probably be ascribed to the lower body weights. No other significant exposure related differences were registered in the preweaning period. Beginning at the age of 7 weeks, male and female offspring were tested for spatial navigation ability in the Morris water maze, where they were required to locate a hidden underwater platform using distal extramaze cues. The performance in the learning situation was similar in the two groups. However, when the test was repeated 4 weeks later, the exposed males had an increased path length (p<0.05). At the same time the swim speed of this group were higher, which probably explains that no differences in latency to locate the platform were registered. In the more difficult task with the hidden platform in a new position, no differences were observed in the males, but the exposed females used more time to locate the platform (p<0.01), probably related to increased path lengths in this group (p<0.01), as the seemingly higher swimming speed of the exposed females was not significant. These results indicate that prenatal toluene exposure may cause a lasting impairment of higher cognitive functions related to spatial learning and memory.

P-85 BRAIN GLIAL FIBRILLARY ACIDIC PROTEIN (GFAP) AND LOCOMOTOR BEHAVIOR AS MARKERS OF NEUROTOXICITY FROM INHALATION EXPOSURE TO TOLUENE Little, A., Gong, Z., Singh, U., Jortner, B.S.*, El-Fawal, H., Evans, H.L. Nelson Institute of Environmental Medicine, New York University Medical Center, Tuxedo, *Virginia Polytechnical Institute, Blacksburg, VA, USA

The concentration of glial fibrillary acidic protein (GFAP) in the rat's brain may be a practical biomarker of toluene-induced neurotoxicity. In order to determine whether GFAP is consistently affected by repeated inhalation exposure to toluene, the concentration of GFAP was measured by ELISA in 4 brain regions during and after sub-chronic exposure to toluene. Adult male rats received inhalation exposure to air or 100, 300, 1000 or 3000 ppm toluene, 6 hr/day, 5 days/wk for up to 42 days. GFAP and locomotion were sensitive to toluene in concentrations that are near the threshold limit value. GFAP and locomotion were affected by toluene concentrations which did not produce overt signs of neurotoxicity or affect body weight or neuronal histopathology (H&E stain at the light microscopic level). Neurological signs and weight loss appeared only after 3,000 ppm. Serum corticosterone was elevated in rats exhibiting early changes in brain GFAP. More research is needed to identify mechanisms which determine whether the toluene-induced changes are an increase or a decrease in brain GFAP concentration. For toxicity screening of inhaled solvents, a battery of GFAP, behavioral and neurotransmitter measures would be useful. Supported by an Environmental Health Sciences Center (ES-00260) and by the American Petroleum Institute.

P-86 QUALITATIVE STARTLE REFLEX ASSESSMENT FAILED TO DETECT TOLUENE INDUCED HEARING LOSS IN RATS Lund, S.P., Hass, U., Johnson, A.-C.*, Nylén, P.* National Institute of Occupational Health, Copenhagen, Denmark, *National Institute for Working Life, Solna, Sweden

The ototoxicity of medical drugs, organic solvents, metals, and asphyxiants has been demonstrated in animal experiments, but the only test of hearing required within the OECD-guideline system for toxicity testing is the qualitative assessment of the startle reflex included in the Functional Observational Battery (FOB). To evaluate the sensitivity of this FOB-test, 5 groups of 12 rats were exposed either 0, 1000, 1500, 2000, or 3000 ppm toluene 8 hours a day for 10 days. Hearing thresholds were determined at 4, 8, 16, 20 and 32 kHz by Auditory Brain stem Responses (ABRs) to 25-95 dBlin SPL tone-pips in 10 dB steps. The hearing thresholds were determined prior to the toluene exposure, and two weeks as well as three months after the end of exposure. The testing of the FOB, including the qualitative assessment of the startle response to a 115 dBlin SPL "click"-sound, were performed prior to the last measurement of the ABR. The group exposed to 2000 ppm toluene showed permanent threshold shift (PTS) at all the four lower frequencies, the mean PTS ranging from 10 dB at 4 kHz to 35 dB at 16 kHz, but showed no change in the scores of the startle reflex. The group exposed to 3000 ppm toluene showed PTS at all the frequencies tested, the mean PTS ranging from of 35 dB at 32 kHz to 55 dB at 8 and 16 kHz. The mean score of the startle reflex showed no difference from the control group, but the scores differed significantly from the control group (p<0.05) due to a greater variance. These results demonstrate that rats may have a hearing loss of more than 70 dB without change in the score of the startle reflex.

P-87 THE ACUTE EFFECTS OF SHORT-TERM INHALATORY EXPOSURE TO MIXED XYLENES ON DELAYED SPATIAL ALTERNATION IN THE RAT Kulig, B.M., Lammers, J.H.C.M. Dept. of Neurotoxicology and Reproduction Toxicology, TNO Nutrition and Food Research Institute, Zeist, The Netherlands

The objective of the present study was to develop a protocol for the evaluation of effects of inhalatory exposure to organic solvents on short-term memory in the rat using mixed xylenes (XYL) as a model compound. Prior to exposure, adult male rats were trained using operant chambers equipped with two stimulus lights, two levers, a recessed water dipper and baffles located on either side of the water delivery opening. Animals were trained on a discrete-trial delayed alternation (DSA) task using a self-correction procedure. The rat's task was to alternate responding between the right and left lever on each trial in order to obtain water reward. Daily sessions consisted of 90 trials separated by 5, 10, and 20 s delays. When performance had stabilized, rats were exposed to XYL vapor at 0, ~100 ppm, ~350 ppm, or ~1225 ppm for 18 hours/day for 3 consecutive days and testing was conducted immediately following termination of each daily exposure. In addition, separate toxicokinetic studies examining XYL blood concentrations were also carried out. Prior to exposure, accuracy of DSA performance in all groups ranged from ~75 to 90% depending on the length of the delay. Following exposure on Day 1, accuracy was severely affected with responding in the highest dose group dropping to chance levels at all delays. XYL also produced significant increases in psychomotor slowing with trial response latencies showing a two-fold increase in the 350 and 1225 ppm groups. In addition, there was an attenuation of XYL effects on both speed and accuracy end-points across the 3 exposure days which paralleled decreases in XYL blood concentrations. Taken together, these results indicate that the operant DSA protocol utilized may be useful for studying the cognitive effects of inhalatory solvent exposure at the animal level.

P-88 EFFECT OF REPEATED EXPOSURE TO XYLENE ON THE ELECTRO-RETINOGRAM OF RATS Simonsen, L., Beltoft, V., Hass, U., Lund, S.P. National Institute of Occupational Health, Copenhagen, Denmark

The electrophysiological activity of the retina is easily measured in both humans and animals with contact lens electrodes. The recorded waveform, the electroretinogram (ERG), is similar between species and changes in the waveform may be used as a sensitive measure for neurotoxicity in general. Rats (n=12) were exposed to 2000 ppm xylene 6 h pr day for 28 consecutive days. The effect of the exposure on the waveform of the flash evoked ERG was compared with a control group (n=12) two weeks and two months after the cessation of the exposure. Two weeks after the exposure there were significant differences in the ERG's between the two groups. Manifest as differences in the latency of the b-wave at 0.0001 joule flash intensity, and difference in the p-p amplitude of the oscillatory component of the ERG at 10 Joule flash intensity. The difference between the two groups was more pronounced when compared again two months after cessation of exposure, both latency and amplitude of the b-wave were different at several flash intensities and the latency of both a-wave and the oscillatory component were affected. Thus, xylene induced changes may progress further over a long-term period after cessation of exposure.

P-89 EVALUATION BY BEHAVIOURAL TESTS OF TETRACHLOROETHYLENE, ETHYLBENZENE AND N-HEXANE DEPRESSIVE ACTION ON CNS IN RATS AFTER ACUTE INTRAPERITONEAL EXPOSURE Miranowicz-Dzierzawska, K., Skowron, J., Puchalska, H., Patelska, B., Zapór, L. Central Institute for Labour Protection, Warsaw, Poland

The depressive action of 1,1,2,2-tetrachloroethylene, ethylbenzene and n-hexane on CNS has been evaluated after injections into Wistar rats. The behavioural tests included: "open field" describing cognitive activity and emotional response (expressed by the number of squares traversed, rearing, preening frequency, etc.), rotarod performance (the ability of rats to remain on a rotating rod as an index of the neuromuscular function), "hot plate" testing antinociceptive activities and treadmill as an index of the fatigue threshold. Organic solvents were administrered intraperitoneally in single doses equivalent to 1 /4 of their lowest lethal doses (LDLo). The results suggest that tetrachloroethylene and n-hexane have depressive action on CNS in acute exposure bringing equilibrium impairment and physical efficiency handicap. Only tetrachloroethylene raises the pain threshold. Ethylbenzene is the weakest, causing physical efficiency impairment only. The effects were generally reversible - they retracted 24 hours after exposure.

P-90 NEUROPHYSIOLOGICAL EFFECTS OF LONG-TERM EXPOSURE TO A MIXTURE OF ORGANIC SOLVENTS USED IN THE PAINT AND VERNISH PRODUCTION Sinczuk-Walczak, Halina The Nofer Institute of Occupational Medicine, Lódz, Poland

The aim of this work was to examine the nervous system of workers chronically exposed to mixtures of organic solvent at concentrations within or slightly exceeding the MAC values, used in the manufacture of paints and lacquers. The tests were performed on a group of 175 people, 107 men aged 22-59 ( X = 41,25), and 68 women aged 20-55 (X = 38.62). The period of employment was X =17.34 years and cumulative dose index 16.97 for males; for females, the corresponding values were X = 14.75 and X = 11.42, respectively. The control group included 175 people (107 men and 68 women) not exposed to solvents. The evaluation included neurological examination, recording of the electrical activity of the brain (electroencephalography EEG) and examination of visual evoked potentials (VEPs). The neurological investigations revealed more frequent occurrence of such subjective symptoms as headaches, sleep disorders, increased sleepiness during the day, mutability of the mood among the exposed workers than among the controls. Neurologic examinations demonstrated no case of overt disorders of the central or peripheral nervous system which could be caused by occupational exposure. In the EEG findings the most popular abnormalities had the character of generalized and paroxysmal changes. The VEPs were abnormal in 40% of workers. The mean P 100 wave latency was prolonged and mean amplitudes P 100 N2 were reduced. These changes can be interpreted as a subclinical sign of dysfunction of the central nervous system.

P-91 NEUROBEHAVIORAL EFFECTS OF A 28 DAY INHALATION EXPOSURE TO HEMIMELLITENE (1,2,3-TRIMETHYLBENZENE) IN RATS Gralewicz, S., Wiaderna, D., Tomas, T. Nofer's Institute of Occupational Medicine, Lodz, Poland

Hemimellitene (1,2,3-Trimethylbenzene - TMB) is a component of industrial solvent mixtures. In U. S. the TLV value for TMBs has been set at 25 ppm (ACGIH 1996). In the experiment described below effects of a four-week inhalation exposure to TMB vapours (6 h/day, 5 days/week, concentration: 0, 25, 100 or 250 ppm) on behavioural performance were studied starting two weeks after exposure. The following behaviours were assessed: i) seeking water in a radial maze (a test for spatial working memory), ii) spontaneous motor activity in an open-field, iii) passive avoidance learning, iv) paw-lick response to heat after a 2 min painful foot shock (hot-plate test),and v) learning and retention of a two-way active avoidance. No significant effects of exposure on radial maze performance and open-field behaviour were noted at any TMB concentration. In the remaining tests, effects of exposure were well-marked but the concentration-effect relationship was not linear. In rats exposed to TMB at concentration of 25 or 100 ppm, but not in those exposed to 250 ppm of TMB, passive avoidance learning was significantly disturbed. In rats exposed to 100 ppm TMB, acquisition of the active avoidance response was impaired, and the foot shock-induced increase in latency of the paw-lick response to heat was more persistent than in rats exposed to 25 or 250 ppm TMB. In general, in respect of behavioural disturbances as well as the concentration-effect relationship, the effects of exposure to hemimellitene resembled the effects of exposure to another TMB isomer - pseudocumene (Gralewicz et al. Neurotoxicol. Teratol., in press). The results suggest that long-term neurobehavioural disturbances may develop in rats exposed to TMB vapours at concentration as low as 25 ppm.

P-92 STUDY FOR ENZYMEHISTOCHEMISTRY OF INTOXICATED RATS INDUCED BY 1,3-BUTADIENE Zhang, L., Gao, S., Xie, K., Sun, K. Dept. of Toxicology, Shandong Medical University, Jinan, P.R.China

To dissect the enzyme changes of neuromuscular junction and the toxic effects to neruotransmitter exposed to 1,3-butadiene, we determined the activity of cholinesterase (ChE) and succinic dehydrogenase (SDH) of musculus quadriceps femoris, the activity of monoamine oxidase (MAO) of brain and sciatic nerve by enzyme histochemistry .The results showed that the activities of ChE and SDH were increased significantly, swelling and multiplication of chondriosome in rats of exposed group, while the activities of MAO in rat cerebrum, striate body, black substance and sciatic nerve were decreased significantly. The results suggest that 1,3- butadiene probably induces peripheral neuropathy through influencing the quantity of main neurotransmitters by increasing the activities of ChE and MAO degrading acetylcholine and catecholamine, while the soaring of SDH indicated that the energy deposited in the muscule may be overconsumed in the intoxicated groups is partly responsible for the damages. Also the changes of activities of MAO in brain reveal that 1,3-butadiene could induce damages of central nervous system.

P-93 THE EFFECTS OF Ca2+ -ATPase AND Na+ / K+ -ATPase ACTIVITIES IN NERVE CELL INDUCED BY ALLYL CHLORIDE Xie, K., Zhang, L., Gao, S., Sun, K. Institute of Toxicology, Shandong Medical University, Jinan, P.R.China

Using chicken embryo whole brain cell, the allyl chloride-induced changes of Ca2+-ATPase and Na+ /K+ -ATPase activities in the plasma and mitochondrion membranes were studied. The results showed that the activities of Ca2+ -ATPase and Na+ /K+-ATPase in the cytoplasm membrane increased sharply with toxic dosages added. Compared with the control group, both of the toxic groups had significance of differences (P<0.01 ). In the mitochondrial membrane, the activities of Ca2+-ATPase tended to go down with allyl chloride increased. However, the activities of Na+ /K+ -ATPase were opposite and tended to up. The results of enzymatic tissue chemistry also showed that the activities of Ca2+ -ATPase increased. The black sediment in the toxic groups much more than that in the control. These results can suggest that allyl chloride could result in the activity changes of Ca2+ -ATPase and Na+ /K+ ATPase. The changes might be related to the variations of Ca2+, Na + and K+ contents in the cytoplasm and mitochondrion of the nerve cell.

P-94 CHANGES OF ELECTROPHYSIOLOGY AND Na+, K+, Ca2+ CONTENTS INDUCED BY ALLYL CHLORIDE IN NERVE TISSUE Xie, K., Sun, K. , Gao, S., Li, B., Zhang, L. Institute of Toxicology, Shandong Medical University, Jinan, P.R.China

Using the methods of fluorescence molecular probe, electron probe X-ray microanalysis (EPMA) and electrophysiology, the allyl chloride (AC)-induced changes of main ions ( Na+ ,K+ ,Ca2+) in nerve cells in vitro and nerve axon in vivo, resting membrane potential (RMP) in NG108-15 cell line, compound action potential (CAP) in rat sciatic nerve, and action potential inside perineural sheath (APIPS) in mice intercostal nerve, were investigated. The results showed that as AC added, [Ca2+]i and [Na+]i were increased markedly in nerve cells; Ca2+ increased and K+ decreased significantly, but Na+ just increased slightly in rat sciatic nerve axon; RMP became smaller; CAP fell down; APIPS showed the waveform of K+ outward current disappeared gradually. The results reveal that the changes of RMP, CAP and APIPS induced by AC are associated with the variations of Na+ ,K+ and Ca2+ in nerve cell and nerve axon, and indicate that the lowered concentration differences of Na+ and K+ between inside and outside nerve axon and increased Ca2+ in nerve axon might further result in the axon degeneration and the depression of excitation and conduction.

P-95 THE MECHANISM OF ALLYL CHLORIDE-INDUCED CYTOSKELETAL INJURY IN NERVE CELLS Xie, K., Sun, K., Gao, S., Zhang, L. Institute of Toxicology, Shandong Medical University, Jinan, P.R.China

To dissect further the molecular mechanism of toxic neuropathy induced by allyl chloride(AC), we used fluorescence molecular probe (Fura-2/AM), the electron probe X-ray microprobe analysis (EPMA) and biochemical methods to determine cytosolic free Ca2+ concentration, the contents of intracellular Ca2+ percentage and Ca2+-free CaM, the activity of Ca2+ /CaM-PK II (Ca2+ /CaM-dependent protein kinase II), and cytoskeleton protein synthesis in chicken embryo brain cell after treated by AC. The results showed that with AC added, the contents of Ca2+ percentage and the concentrations of cytosolic free calcium, and the activities of Ca2+/CaM-PK II in cells were increased significantly (P<0.01), however, the contents of Ca2+-free CaM and cytoskeleton protein synthesis were markedly decreased (P<0.01). The results suggest that one of the mechanism of AC-induced cytoskeleton injury in vitro might be related to elevation of intracellular Ca2+,activated CaM and Ca2+/CaM-PK II.