Invited Speakers:

THE PON1 GENE AND DETOXIFICATION

Furlong, CE*; Li, W-F*;. Richter, RJ*; Shih, DM**; Lusis, AJ **; Costa, LG*, *University of Washington, Seattle;**University of California at Los Angeles, USA.

It has been assumed since its discovery that serum paraoxonase (PON1) plays a major role in the detoxication of specific organophosphates. It was also assumed that individuals with low PON1 activity would be more susceptible to paraoxon/parathion poisoning than individuals with higher PON1 activity. Evidence supporting this hypothesis was provided by injection of rabbit PON1 into rodents. Injected PON1 protected against paraoxon toxicity in rats and chlorpyrifos oxon toxicity in mice. The recent availability of PON1 knockout mice has provided an in vivo system with which to more closely examine the role of PON1 in detoxication. PON1 knockout mice demonstrated dramatically increased sensitivity to chlorpyrifos oxon and diazoxon and moderately increased sensitivity to the respective parent compounds. The PON1 knockout mutation also resulted in the elimination of liver PON1 activity, accounting for the dramatic increase in sensitivity to chlorpyrifos oxon and diazoxon. Totally unexpected was our finding that the PON1 knockout mice were not more sensitive to paraoxon. This was particularly surprising in light of the earlier enzyme injection experiments. Differences in the relative catalytic efficiencies of rabbit vs. mouse PON1 for the specific oxon forms explain these observations. Mouse PON1 has good catalytic efficiency for the hydrolysis of diazoxon and chlorpyrifos oxon, but a poor efficiency for paraoxon hydrolysis relative to rabbit PON1. The human PON1_Q192 isoform has a catalytic efficiency similar to that of mice, whereas the human PON1_R192 isoform has a much better catalytic efficiency, predicting that individuals expressing high levels of the PON1_R192 isoform should have increased resistance to paraoxon toxicity.

Supported by NIH Grant P01HL30568; NIEHS Center Grant P30 ES 07033; and UW Center Grant for Child Environmental Health Risks Research (NIEHS 1 PO1 ES09601-01/EPA-R826886-01-0).
 

NTE: NEW INSIGHTS INTO ITS STRUCTURE, PHYSIOLOGICAL FUNCTION AND ROLE IN ORGANOPHOSHORUS-NEUROPATHY.

Paul Glynn, MRC Toxicology Unit, University of Leicester, UK.

Neuropathy Target Esterase (NTE) is an integral membrane protein in neurons, originally identified as the primary target for those organophosphorus esters (OP) which cause delayed neuropathy (OPIDN). NTE has serine esterase activity and efficiently hydrolyses phenyl valerate (PV) in vitro, but its physiological substrate is unknown. NTE is the vertebrate homologue of the Swiss Cheese protein (SWS) in Drosophila which regulates interactions between neurons and glia in the developing nervous system. The sws mutation leads to widespread neurodegeneration but, in adult vertebrates NTE appears to be redundant. The key biochemical lesion initiating OPIDN is the generation of a negatively charged adduct at NTE’s active site. We suggest that this may cause a toxic gain of function in NTE and that one of the factors which render an animal species susceptible to OPIDN is the presence of a sufficiently high level of NTE.  Analysis of NTE’s primary sequence predicts four transmembrane segments (TM#1-4) and suggests it comprises an N-terminal regulatory domain and a C-terminal effector domain. The active site serine of NTE resides in this C-terminal domain at the centre of predicted TM#4. Therefore, to achieve PV hydrolysis, TM#4 must line an aqueous transmembrane pore. We have incorporated the purified recombinant effector domain of NTE into giant liposomes and shown by patch-clamp experiments that it forms a transmembrane pore with a substantial conductance.
 

CHARACTERISATION OF PROMOTION TARGETS

Moretto A, Istituto di Medicina del Lavoro, Università di Padova, Padua, Italy

Certain esterase inhibitors including organophosphates, organophosphinates, sulfonyl halides, carbamates and thiocarbamates exacerbate the clinical and morphological expression of toxic and traumatic axonopathies (promotion). The model axonopathy used to characterise promotion is organophosphate induced delayed polyneuropathy (OPIDP). The target of OPIDP is a neural phenyl valerate esterase (PVE) called neuropathy target esterase (NTE), defined as the PVE resistant to paraoxon and sensitive to mipafox (40 and 50 µM, respectively). Promotion is neither a toxicokinetic interaction (promoters do not modify NTE inhibition by the neuropathic compound) nor an additive phenomenon (most promoters are not neuropathic). There are indications that the target of promotion might be similar to NTE. Paraoxon-resistant PVEs were titrated with mipafox: besides NTE, another PVE was identified with an approximate I₅₀ of 200 µM (M200) and operationally defined as the PVE resistant to paraoxon and mipafox (40 µM and 50µM, respectively) and sensitive to paraoxon and mipafox (1 mM). The I₅₀ for the promoters PMSF (about 100 µM) and butylsulfonyl fluoride (about 60 µM) were similar for both NTE and M200. On the contrary, M200 I₅₀s for the neuropathic compounds diisopropylfluorophosphate (DFP) and dibutyldichlorvos were much higher than those for NTE (2-10 and 0.06-0.2 vs. 0.2-0.5 and 0.004-0.008 µM, respectively). M200 and NTE were selectively inhibited in vivo by the promoter KBR 2822 (O-(2-chloro-2,3,-trifluorocyclobutyl) O-ethyl S-propyl ester) and by DFP, respectively. A high and a low molecular weight mipafox-sensitive PVE have been identified by means of chromatography in the supernatant of peripheral nerve. The former resembled NTE and was inhibited after treatment with DFP (0.5 mg/kg sc). The latter (about 60 KDa) was reversibly inhibited by paraoxon in vitro and by KBR 2822 in vivo (2.5 mg/kg po). This suggested that M200 might correspond to the low molecular weight activity and its inhibition correlates with promotion.
 

THE CHRONIC EFFECTS IN HUMANS OF EXPOSURE TO ORGANOPHOSPHATE PRODUCTS

Pilkington A*, Buchanan D*, Jamal GA**, Hansen S***,Gilham R***, Kidd M*, Hurley JF*, Soutar*, *Institute of Occupational Medicine, **Division of Neuroscience, Imperial College, London, ***Institute of Neurological Sciences, Glasgow.

This study, with three distinct phases, aims to investigate the hypothesis that repeated exposures to organophosphate pesticides (OPs) may cause cumulative and irreversible damage to nervous tissue, which becomes sufficient to be clinically detectable.
The first phase of the study was completed in September 1996 and sought to develop a model for uptake of OPs based on observation of a retrospective exposure history questionnaire used in the second phase of the project.
The second phase cross-sectional field study of 600 farm personnel and 200 individuals with low exposure to OPs (chicken and pig farmers and ceramics workers) was completed in June 1997. Data were collected on exposure history, neurological symptoms and results of standardised tests of vibration and thermal sensation. The second phase aims to examine the relationship between cumulative history of exposure to OPs and clinically relevant indices of peripheral neuropathy. The third phase clinical studies involved detailed neurological and neuropsychological investigations of a subgroup of individuals from the cross-sectional study. This phase sought to validate the results obtained in the field and included more sophisticated neuropsychological and neurophysiological tests which could not be performed in a field setting.
Data analysis is ongoing and a report is currently being prepared for the sponsors. The project completion data is 30 April 1999.
 

GENE TARGETING, THE HIPPOCAMPUS, AND NEUROTOXICITY

R. Lathe. Centres for Genome Research and Neuroscience, University of Edinburgh, King's Buildings, Edinburgh EH9 3JQ, UK (rlathe@ed.ac.uk).

Susceptibility to toxic insult is modulated by pathways of metabolism and delivery, and gene modification in mouse embryonal stem cells (ES cells) has been used to investigate the roles of individual components, illustrated by `knockouts' for genes encoding multidrug resistance proteins, glutathione S-transferases, glutathione peroxidase, and cytochromes P450. The brain, for reasons unknown, is highly prone to insult, but new molecular evidence, including data from random gene targeting, may indicate two reasons for the particular susceptibility of one brain region, the hippocampus.
Analysis of functional insertions of a promoter-less cassette into ES cells ('gene-trapping') suggested an unexpectedly high frequency of gene expression in the hippocampus. In the 3 cases where gene cloning was performed these encoded receptors and signalling molecules; this prompted a general study that revealed unusually diverse and robust receptor expression in the hippocampus. It is inferred that a primary role of the hippocampus is in sensing of ligands that reflect the internal status of the body (enteroception), that may reflect a shared origin with the olfactory system (cf. exteroception). Abundant receptor expression may predispose the hippocampus to toxic damage.
In addition, the hippocampal formation is itself a site of oxidative metabolism, and one cytochrome P450, Cyp7b, was found to be fairly selectively expressed in this brain region. Cyp7b could modulate local neurotoxicity through toxin activation/deactivation, but this has not yet been demonstrated. However, Cyp7b oxidises a number of substrates including the major adrenal steroid dehydroepiandrosterone (DHEA), and the major product (7alpha-hydroxyDHEA) appears to inhibit glucocorticoid action. Cyp7b may thus be neuroprotective because excess glucocorticoids can predispose to neuronal damage. Targeted disruption of the Cyp7b gene will be discussed.
Finally, because the hippocampus can govern many aspects of body physiology, including but not restricted to HPA axis activity, brain effects mediated by the hippocampus may dictate some physiological signs of 'systemic' toxicity.
 

GENETIC DISSECTION OF KEY STEPS IN NEUROTRANSMITTER RELEASE

Augustin, I.*; Rosenmund, C.**; Sudhof, T.C.***; Brose, N.*; *Max-Planck-Institut fur Experimentelle Medizin, Gottingen, Bundesrepublik Deutschland ; **Max-Planck-Institut fur Biophysikalische Chemie, Gottingen, Bundesrepublik Deutschland; ***Center for Basic Neuroscience, Howard Hughes, Medical Institute, University of Texas Southwestern Medical Center, Dallas, TX, USA.

Neurotransmitter release at synapses between nerve cells is mediated by Ca²⁺-triggered exocytotic fusion of synaptic vesicles. Before fusion, vesicles dock at the presynaptic release site. A priming process of unknown molecular nature is thought to cause the maturation of docked vesicles to a fusion-competent state. Using deletion mutant mice, we identified munc13-1, a presynaptic phorbol ester receptor, as an essential synaptic vesicle priming factor. Glutamatergic hippocampal nerve cells from mice lacking munc13-1 form ultrastructurally normal synapses in which docked vesicles are not primed and thus unable to fuse with the plasma membrane in response to physiological stimuli. In contrast, GABAergic neurons are not affected by the lack of munc13-1, indicating the existence of transmitter-specific priming processes in synapses. This essential role of munc13-1 in synaptic vesicle priming may be mediated via its interaction with syntaxin, a target of chlostridial neurotoxins involved in synaptic vesicle exocytosis.
 

EARLY RESPONSE GENE MARKERS OF NEUROTOXICITY

Kind, CN. Safety Assessment, AstraZeneca R&D Charnwood, Loughborough, UK.

Early gene expression plays a key role in the cellular response to injury in the CNS and other tissues. A variety of genes, including those encoding for stress proteins and transcriptional regulators are important determinants of protective and adaptive mechanisms mounted by a cell in response to the initiating lesion.
Hsp72 is a stress-inducible member of the ubiquitous Hsp70 heat shock protein family whose expression is up-regulated in response to a wide range of noxious physical and chemical agents. Hsp72 appears to play a protective role within the injured cell by facilitating the disposal of damaged proteins and stabilising the conformation of newly synthesised ones.
Nuclear transcription factors are important in the regulation of short and long term responses to external stimuli, including both physiological events and pathological processes. Thus, long-term potentiation of memory, neuronal development and the response to lesions and chemically-induced seizures have all been shown to involve the induction of c-fos and c-jun. The ubiquitous heterodimeric nuclear transcription factor NF-kB is activated by a number of agents including a variety of cytokines. Although it’s role in immune and inflammatory responses of the CNS is well established, it may also be involved in a number of other processes.
Acute exposure of rats to MK 801 (dizocilpine maleate), a potent non-competitive N-methyl-D-aspartate receptor antagonist, produces a variety of biochemical, physiological and pathological changes in the brain , including temporary vacuolation, and at high doses, necrosis in a sub-population of neurones in the cingulate cortex. Studies of early stress gene responses following MK 801 administration have been useful in characterisation of the lesion produced. These studies will be described and particular emphasis placed on the role and utility of measuring early response genes in neurotoxicity.
 

SILVER DEGENERATION STAINS REVISITED IN LIGHT OF GLIAL REACTIONS TO NEUROTOXIC INJURY

Fix, AS. Procter and Gamble, Miami Valley Laboratories, Cincinnati, Ohio, 45253, USA

Detecting morphologic evidence for neurotoxicity is a challenge for those interested in locating and interpreting the significance of injury in the brain. Although detailed morphologic examinations using specialized techniques can be made in the academic research setting, screening studies required for neurotoxicology safety evaluation by industrial toxicology laboratories do not lend themselves readily to similar detailed evaluations. The present work revisits the potential application of silver degeneration stains for broader use in neurotoxicology. In addition, silver stain data is interpreted in light of glial cell reactions to injury, specifically glial fibrillary acidic protein (GFAP) staining for astrocytes and histocompatibility antigens (MHCs)/complement receptor (CR) staining for microglia. Neurotoxic injury induced by the NMDA antagonist MK-801 (dizocilpine maleate), which produces neuronal cell death in the cerebral cortex of the rat, was studied by the cupric-silver degeneration stain in composite frozen sections. Cupric-silver staining revealed affected nerve cell bodies, dendrites, and axons in a highly specific manner. GFAP immunohistochemistry showed localized and time-dependent astrocyte reactivity in the area of cell body injury. Histochemistry (Griffonia simplicifolia lectin) and immunohistochemistry for CR (OX-42), MHC 1 (OX-18), and MHC 2 (0X-6) showed phenotypic change by microglia, as well as upregulation of MHC 1 and 2. Phenotypic reactivity of microglia was confined to the area of nerve cell body injury, while antigen upregulation occurred there as well as in more distant areas containing dendritic and axonal damage. This work shows how the concurrent morphologic evaluation of silver stains for neuronal degeneration and special immunohistochemical stains for glial cells enhances our ability to detect and interpret neurotoxic injury in the brain.
 

FACTORS CONTROLLING THE BALANCE BETWEEN APOPTOTIC AND NECROTIC MODES OF NEURONAL DEATH.

P. Nicotera, Faculty of Biology, University of Konstanz, Box 5560-X911 D-78434, Konstanz, Germany

Apoptosis and necrosis, in their characteristic appearances, are clearly distinct modes of cell death. However, a large body of evidence suggests that these two may just represent extremes of a potentially continuous spectrum of possibilities for a cell population to die. In tissues, apoptosis and necrosis may either coexist or be sequential events, and the mode of cell death may be influenced by the intensity or exposure time of a stressful event. We have shown that the degree of mitochondrial dysfunction and the cellular ATP level are critical factors that decide the mode of cell death. The mode of cell demise, apoptosis or necrosis, may be relevant to decide the onset and/or progression of neuroinflammation. Endogenous mediators, including NO, cytokines or certain classes of chemokines can interfere with the execution of apoptosis and thereby decide the prevalent shape of cell death. These mediators or local metabolic conditions (i.e., low energy state) may interrupt execution of apoptosis before recognition molecules for phagocytosis appear on the cell surface. This would result in inefficient removal of "undead" cells and in delayed cell lysis, with release of pro-inflammatory molecules. Such condition would favour neuroinflammation, perpetuate neuronal damage and may explain the characteristic slow progression of neurodegenerative diseases. Thus, therapies that aim to prevent apoptosis may not be beneficial in all cases, and while in some instances they effectively protect from tissue damage, in others may favour persistence of undamaged cells and stimulate inflammation.
 

PREDICTION OF NEUROTOXICITY BY MOLECULAR TARGET MODELLING; THE CHALLENGE OF POST-GENOMIC TOXICOLOGY

L.L.Iversen, Department of Pharmacology, University of Oxford, Mansfield Road, Oxford, UK

Molecular genetics research is revealing the remarkable diversity of ion channels and cell surface receptors that exist in vertebrate and invertebrate species. Completion of the analysis of the C.elegans genome, for example, has revealed the existence of more than 30 different potassium channels, many of which had not hitherto been described. This is remarkable for an organism that possesses only 302 neurones. Several of the potassium channel genes are expressed only in a single type of neurone, and some in only a single neurone; they could not have been discovered by conventional means. More than 1000 genes encode G-protein-coupled cell surface receptors, many of which are involved in chemosensory function.
Analysis of the human genome is likely to reveal a similar abundance of hitherto unknown ion channels and cell surface receptors, that can act both as therapeutic targets or as the origins of neurotoxic reactions to novel drugs. The ligand-gated ion channel receptors for the amino acid neurotransmitters GABA and L-glutamate, for example, offer great diversity and many opportunities for further discoveries. The so-called "orphan" nuclear receptors for steroid-like molecules, many of which are expressed in the nervous system, represent another challenge for neurobiology that has been revealed by genomic research.
 

ENGINEERING NOVEL MINIPROTEINS OUT OF TOXINS?

Ménez, A. and Vita, C. DIEP, CEA/CE Saclay, 91191 – Gif sur Yvette Cedex, France.

Potent toxic proteins are produced by a variety of venomous animals from various phyla. They are often of small size, possess a large density of disulfide bonds and exert multiple functions directed toward various targets, including a diversity of enzymes and ion channels. The structural basis for the diversity of toxin functions is becoming understood. Thus, a toxin fold can exert numerous unrelated toxic functions and unrelated protein folds can exert similar toxic functions. Furthermore, evidence is now accumulating that any region of a toxin fold can display a functional "toxic" topography. Therefore, a toxin fold is functionally highly permissive. We tentatively exploited this property to force a toxin fold to exert non-toxic and potentially useful biological activities.
The fold of scorpion toxins is well suited to engineer novel miniproteins. It comprises 30-40 residues and includes a ß–hairpin linked to a short helix by 3 disulfide bonds. Among various constructions, we succeeded in reproducing, on the scorpion toxin fold, the core region by which the human CD4 surface interacts with the HIV-1 envelope glycoprotein. Eight solvent-exposed residues from a ß-hairpin of the human CD4 that are central in binding to gp120 were grafted to a structurally homologous ß-hairpin of the scorpion toxin scaffold. The resulting chimera loose its original toxic properties, but became capable of binding specifically to gp120 with an IC₅₀ of 40 µM. The structural and functional reproduction of the grafted residues seemed quite good, as judged from NMR and mutational analyses of the chimera. Introduction of five additional mutations led to a miniCD4 that not only bound gp120 with a 100-fold higher affinity but also prevented infection of CD4⁺ cells with several HIV-1 isolates. The miniCD4 may be interesting to study how the interaction between CD4 and gp120 favors entry of HIV-1 into cells.
Animal toxins, therefore, constitute a bank of small and highly stable folds that may be engineered to express a diversity of non-toxic functions.
 

MODULATION OF PATTERNED CELLULAR-ELECTRICAL ACTIVITIES AS A PREDICTOR OF NEUROTOXIC VULNERABILITIES

E. C. Conley, Centre for Mechanisms of Human Toxicity, University of Leicester, Lancaster Road, Leicester, U.K.

An extraordinary diversity of patterned electrical (ionic) signalling has been associated with differentiated functions of specified neurons and glia within the nervous system. The principal molecular 'switches' effecting these are the ion channels. Vertebrate genomes encode several hundred ion channel subtypes, each of which has evolved to perform a highly-selective electrical 'task' within cells. These 'tasks' can be discerned by identifying cellular conditions which 'gate' (open/close) the channel, together with the 'modulatory' responses to exogenous agents. Physiological gating most often occurs in response to an extracellular or intracellular ligand, a channel protein phosphorylation or a change in transmembrane voltage. Any of these primary responses can be inhibited or enhanced by toxic agents.
Protein expression mapping systems for ion channels are being developed that aim to link 'expression topography' of ion channels and receptors in the CNS with their functional significance. These frameworks could aid neurotoxicology by providing tools to co-map lesion vulnerabilities and target protein distributions in internet-accessible archives. Protein level, evolutionary, structural and functional similarities across apparently dissimilar ion channel and receptor subtypes may enable us to predict common vulnerability to drug side-effects and toxicity based on shared modulatory motifs. The prediction of effects of toxicants on specific gene family members remains a difficult problem. One potentially useful model (linking ion channel isoform specificity with cell function) uses information on ligand, phosphorylation or voltage sensitivities of components to suggest a likely order of gating events (from rest) amongst multiple co-expressed channel subtypes. Such functional multiprotein 'machines' may cycle spontaneously through a highly-stable succession of intrinsic gating events (as in 'pacemaker' neurons) or may be assembled dynamically in 'resting' cells following receptor stimulation (as in many types of neurosecretion). Thus, by determining how toxicants modulate receptor, transducer and/or effector protein activities within cell-type excitability cycles, one may be able to develop a predictive framework for functional vulnerabilities induced by agents
 

DEVELOPMENTAL EXPOSURE TO PCB CONGENERS AND DEFINED MIXTURES - ENDOCRINE AND NEUROBEHAVIORAL EFFECTS¹

Lilienthal, H*; Hany, J*, Kaya, H*, Roth-Haerer, A*, Sarasin, A**; Lichtensteiger, W**; Winneke, G*. *Medical Institute of Environmental Hygiene, Duesseldorf, Germany; **Institute of Pharmacology, University of Zuerich, Zuerich, Switzerland.

Polychlorinated biphenyls (PCBs) form a class of wide-spread environmental contaminants which accumulate in food chains and are found in breast milk at elevated levels. The developing nervous system is particularly sensitive to PCB exposure. Gestational exposure of rats to a non-ortho-chlorinated, coplanar PCB congener led to changes in haloperidol-induced catalepsy and passive avoidance in adulthood. In addition, the distribution of activity in the open field was altered as rats treated with the coplanar PCB exhibited more activity in the inner zone than controls. Moreover, increases in conditioned ultrasonic vocalizations were found in another experiment using maternal exposure to a mixture of PCBs reconstituted according to the congener pattern found in breast milk. Thyroid hormones and steroids are important regulators of neuronal development and PCBs are reported to interact with these hormones. Therefore, developmental PCB effects on the nervous system might be mediated by influences on hormone-dependent processes. For instance, sexual differentiation of the brain is dependent on aromatase (CYP19) which converts androgens to estrogens. Estradiol has a prominent role in neuronal plasticity and induces a male-like development of brain and behavior. Aromatase activity was determined in newborn male pups and found to be decreased by maternal exposure to the reconstituted PCB-mixture. Adult male littermates exhibited lower testes weights and reduced testosterone concentrations together with a higher, more female-like sweet preference. Finally, a place preference test detected a higher stimulation by testosterone in maternally exposed male rats.

¹supported in part by the Department of Interior, State of Baden-Wuerttemberg, Germany, grants PUG 95003 and PUG 97004 to H.L.
 

BEHAVIORAL EFFECTS OF DEVELOPMENTAL EXPOSURE TO FISH-BORNE CONTAMINANTS

Joseph L. Jacobson, Dept., of Psychology, Wayne State University, 71 W Warren Ave., Detroit, MI 48202, USA.

Polychlorinated biphenyls (PCBs), man-made compounds once widely used in electrical transformers and capacitors, are among the most ubiquitous and persistent environmental contaminants. Although banned in most western nations since the 1970’s, PCBs continue to be found in a variety of foods, including fish, cheese, and fatty meats. 313 infants delivered in western Michigan hospitals in 1980-81 were recruited to overrepresent those whose mothers had eaten relatively large quantities of PCB-contaminated Lake Michigan fish. Prenatal exposure to these compounds was assessed in terms of umbilical cord blood and maternal blood and milk samples obtained shortly after delivery. Although recruited to overrepresent a heavily exposed population, the average maternal PCB body burden was not exceptionally high and fell at the upper end of general population levels. The children’s intellectual function was assessed during infancy and at ages 4 and 11 years. A broad range of potential confounding variables was assessed and controlled statistically in the data analyses. Prenatal PCB exposure was associated with poorer short term memory and attentional function at all three ages. At 11 years, the IQ scores of the most heavily exposed children averaged 6.2 points lower than the others in the sample, and these children lagged in reading comprehension by an average of 7.2 months. Although much larger quantities of PCBs were transferred via lactation to the infants who breast-fed, deficits were seen only in relation to transplacental exposure, suggesting that the developing fetal brain is particularly sensitive to these compounds. Environmental concentrations of PCBs have declined in the U.S. in recent years, but the risk of exposure from toxic industrial waste continues due to the large quantities of these compounds still in use in older electrical equipment and trapped in landfills.
 

EFFECTS OF ENVIRONMENTAL EXPOSURE TO POLYCHLORINATED BIPHENYLS AND DIOXINS ON COGNITIVE DEVELOPMENT IN YOUNG CHILDREN

Hestien Vreugdenhil, Nynke Weisglas-Kuperus, Sophia Children's Hospital, Erasmus University Rotterdam, Department of Paediatrics, Division of Neonatology; P.O. Box 2060, 3000 CB Rotterdam, The Netherlands.

In the Netherlands a prospective study was started in 1989 to investigate the effects of background Polychlorinated Biphenyl (PCB) and dioxin exposure on the development of healthy term born babies. From birth until school age the children are followed in their neurological, cognitive and behavioural development.
The cohort consists of 418 healthy mother infant pairs. 207 pairs were living in Rotterdam, a highly industrialised area in the Netherlands and 211 pairs in Groningen, a semi-urban area in the north. 209 children were breast-fed and 209 were formula-fed during infancy.
Prenatal PCB exposure was estimated from the sum of four PCB congeners, IUPAC numbers 118,138,153,180 in maternal plasma and cord plasma. Lactational exposure was assessed from breast milk PCB and dioxin concentrations, multiplied by the number of weeks of breast-feeding.
At 3, 7, and 18 months development was assessed with the Dutch version of the Bayley Scales of Infant Development, at 42 months the Kaufman Assessment Battery for Children (K-ABC) and at 6 years with the McCarthy Scales of Development. Neurological examination was done at birth, 18 and 42 months and 6 years of age.
Prenatal PCB exposure was related to lower psychomotor scores at 7 months of age, poorer neurological condition at birth and at 18 months. At 42 months prenatal PCB exposure was related to lower scores on the K-ABC, but no effect on the neurological condition was seen. Postnatal PCB and dioxin exposure through lactation was related to lower psychomotor development at 7 months.

This study is funded by the European Community (contract no. EV5V-CT92-0207).
Koopman-Esseboom C. et al. (1996) Pediatrics 97:700-6 4 (2). Huisman M. et al. (1995) Early Hum Dev 41: 111-27 61. (3) Huisman M. et al. (1995) Early Human Development 165-176 805. (4) Patandin S. et al. (1999) J Pediatr 134: .33-41 12.
 

EXPERIMENTAL ANIMAL RESEARCH HAS PROVEN TO BE OF MINIMAL VALUE IN DETERMINING CHEMICAL SAFETY FOR MAN (For the Motion)

Professor Anthony D. Dayan, London

Even without being a dualist, and whilst avoiding the mind-body problem, there is overwhelming evidence and powerful theoretical arguments why human safety has been and largely still is dependent on observations on man. Experiments in animals, both classical whole animal pharmacology and toxicity tests, and the newer transgenic techniques and other manipulated model systems, have proven to be costly delusions diverting thought and money from the specific studies on risks that can only be done in man, or on human tissues ex vivo, that are the sole route to deriving safety from laboratory work.
First, consider the fact that many forms of toxicity, especially those affecting the nervous system and its developed activities, such as cognitive function, intelligence, emotion, mood and memory, and equally those altering other physiological systems and so perverting function, can be detected far more sensitively in humans than in animals, because the latter provide only minimal paradigms restricted to gross abnormalities. They are good for studying marked abnormalities and mechanisms of importance already proven in man, but they cannot provide the sensitivity or breadth of detection that protection of man requires.
Second, as we are so often reminded, there are considerable differences between species in physiology, in metabolism, in toxicokinetics and in the range of individual variation, as well as in voluntary and involuntary exposure to sources of harm. The only way to extract information from the necessarily limited animal studies of new substances or of new effects that can help to protect ourselves is to apply large fudge factors to quantitative findings, and to use the ‘Weight of evidence’ argument, ie ‘We discard these unwanted findings because they don’t fit our preconceived notions’, to jettison qualitative results that we don’t like. The ‘protection’ derived from mechanistic experiments on effects already demonstrated in animals is often no more than post hoc propter hoc argumentation based on simplistic observations and a hopeful belief that all will be well on practical exposure. And, if we do find mechanistic reasons why man and animals may react differently, we have so little confidence in our own hypotheses, that we apply the huge protective factors blindly derived from circumstances where we lack any understanding of why toxicity has occurred.
In short, I suggest that ‘safety’ for humans depends on careful observations on man. What we can learn from ourselves was once obtained from clinical observations and death rates but is now increasingly based on sophisticated physiological, functional and metabolic techniques, ranging from psychometry to metabolic MRI to immunological measures and electrophysiology. They afford sensitive detection systems to find abnormalities and careful means to investigate their causes in ways that will show the dose-response relationships on which true safety assessment alone can be based. Man is indeed the "Real thing" as far as human safety is concerned.
 

EXPERIMENTAL ANIMAL RESEARCH HAS PROVEN TO BE OF MINIMAL VALUE IN DETERMINING CHEMICAL SAFETY FOR MAN (Against the motion)

Marcello Lotti, Università degli Studi di Padova, Istituto di Medicina del Lavoro, via Giustiniani 2, 35128 Padova

The strong social pressure for preventive action against adverse effects of man-made chemicals has shaped responses to it consisting in relatively swift and practical solutions. These are well represented by the current toxicity tests in animals. Such test batteries have been built up over the years and are now evaluated in response to the following question: "What if….?". Judging from the past and actual disputes that accompany these procedures, the whole system is not satisfactory. Therefore, I do concede that a large amount of data obtained from toxicity test in animals speaks in favour of the motion.
Because of this and other reasons, several alternative approaches have been attempted including mathematical modelling, QSARs, in vitro testing, retrospective epidemiology and others. Again, none has proven satisfactory. Therefore, it seems obvious that there are no alternatives to animal research.
However, toxicity tests do not represent the whole spectrum of experimental animal research. On a number of occasions, mechanistic research in animals has proven to be of great value in risk assessment. When molecular targets of toxicity are identified and the cascade of biochemical and physiological reactions following chemical target interactions understood, then extrapolations across species become rational and rewarding. In addition, when safety of chemicals was challenged, or supposed to be, by new evidence including that in humans, then the information gained from mechanistic research in animals was proven to be revealing. Therefore there is an additional or perhaps alternative question to be answered while performing experimental research in animals for the purpose of risk assessment: "How is this…?". In this way we might reduce the number of experiments in animals of dubious significance and concurrently increase the experiments aimed at a better understanding of the mechanisms of toxicity.
Therefore the motion requires rephrasing as follows: "What type of experimental animal research has proven to be of minimal value in determining chemical safety for man?
Certainly, it is not the case of animal research on mechanisms of toxicity, because whenever a paradigm of science changes then enormous practical exploitations follow.
 

NMR SPECTROSCOPY

Bachelard, HS. M.R. Centre, School of Physics and Astronomy, University of Nottingham, Nottingham, NG7 2RD, UK.

Three applications of MR spectroscopy to studies on cerebral metabolic function which may interest neurotoxicologists are briefly reviewed:
1. Excitotoxicity. Glutamate at concentrations above 0.2 mM causes relatively rapid decreases in PCr and ATP (from ³¹P-MRS), accompanied or immediately followed in ¹⁹F-MR spectra by increased free intracellular calcium and release of zinc (possibly neuroprotective). Similar effects are observed with low (10 mM) concentrations of NMDA. Pre-treatment for 1 hr with the NMDA blocker, 10 mM-MK801, prevented all of the observed effects of NMDA. In contrast, MK801 had little detectable effect on the increase in Ca²⁺ or the decreases in PCr and ATP caused by glutamate, though it prevented the release of zinc. These results have implications for current views on the excitotoxic hypothesis of cell death in e. g. stroke¹.
2. Selective cerebellar toxicity of L-2-Chloropropionic acid. This compound is selectively toxic to cerebellar granule cells in the rat some 36-48 h after administration. ¹H- and ³¹P-MRS studies showed that the known activation of the pyruvate dehydrogenase complex (PDH) is non-selective in that it occurs in both cortex and cerebellum within 2 hours, and that these effects have disappeared well before toxic symptoms are seen. Increases in lactate and alanine, and decreases in NAA and the energy state were observed only at the onset of toxic symptoms (severe hindlimb weakness and abnormal gait), and were restricted to the cerebellum², where ¹H-MR images revealed extensive granule cell necrosis, with considerable swelling of ventricular spaces. MK801 caused normalisation of all parameters except that some energy deficit remained. The results indicate that the selective toxicity is not directly related to activation of PDH and may involve glutamate activity through the NMDA receptor.
3. Selective glial toxicity of Fluoroacetate. Much intermediary metabolic information can be derived from isotopomer analysis of ¹³C-MR spectra of brain extracts labelled in vivo from precursors: [1-¹³C] glucose (which labels the CH₂-groups of intermediates such as glutamate, glutamine and GABA) or [1,2-¹³C] acetate (which labels also the COOH groups of these metabolites, but only in glia). Thus the C4,C5 isotopomer of the C4 resonance of glutamine must be derived from acetate, as is the C2,C1 isotopomer of GABA³. Mice were injected with combinations of these precursors in the presence or absence of F-acetate. The C4,C5 isotopomers of glutamine confirmed the specificity of the toxin as an inhibitor of the glial TCA; the neuronal TCA remained intact. However although the glial TCA was inhibited, the glial conversion of glutamine to glutamate was not affected. The C2,C1 isotopomers of GABA revealed that much of the GABA must have been derived from glial glutamine. These results provided unique in vivo confirmation that glial glutamine is an important precursor for neuronal GABA⁴.

1. Thatcher NM; Morris PG; Prior MJW; Bachelard HS (1999) J.Neurochem 72, 2741-2748
2. Williams RE; Jones P; Lock EA; Bachelard HS (1999) J.Neurochem 73, 362-371
3. Taylor A; McLean M; Morris P; Bachelard H (1996) Dev. Neurosci. 18, 434-442.
4. Hassel B; Bachelard H; Fonnum F; Jones P; Sonnewald U (1997) J. Cereb. Blood Flow Metab. 17, 1230-1238.
Supported by the MRC, the Leverhulme Trust and Zeneca CTL
 

USE OF AGGREGATING BRAIN CELL CULTURES TO ADDRESS DEVELOPMENTAL EFFECTS OF ACETYLCHOLINESTERASE INHIBITION BY ORGANOPHOSPHORUS INSECTICIDES.

Monnet-Tschudi, F.*; Zurich, M-G.*; Schilter, B.**; Costa, L.G.*** and Honegger, P.*. *Institute of Physiology, Lausanne Switzerland; **Nestlé Research Center, Lausanne; ***Dept. Environmental Health, University of Washington, Seattle, USA and Dept. of Pharmacology and Physiology, University of Roma"La Sapienza", Roma, Italy.

For many organophosphorus compounds (OP), safety standards are derived from their potential to inhibit acetylcholinesterase (AChE). The adequacy of such an approach to cover all the potential neurotoxicological effects including developmental neurotoxicity has been recently challenged. Aggregating brain cell cultures of fetal rat telencephalon have been used as model to address these issues, using parathion, chlorpyrifos, their respective oxons, and physostigmine as a reversible AChE inhibitor. Cultures were treated with OP either during an early developmental period corresponding to birth till postnatal day 10 or during a more mature stage corresponding to postnatal days 20 to 30. A wide range of concentrations ranging from overtly toxic to inactive were selected. Cell type-specific effects were analyzed with regards to the AChE inhibition. Each compound produced a specific pattern of neuronal and glial effects. These cell type-specific effects were found at concentrations causing more than 80% AChE inhibition. They were more pronouced in immature cultures, suggesting an intrinsic developmental sensitivity of brain cells. Chlorpyrifos, chlorpyrifos oxon and paraoxon caused a selective loss of cholinergic parameters. AChE inhibition was the most sensitive change observed after OP treatments. The oxon forms were 10-100 times more potent AChE inhibitors than the parent compounds.These in vitro data suggest that although toxic effects may differ according to the OP considered, AChE inhibition remains however the most sensitive marker of brain exposure to OP.

This work was supported by Nestlé and by the Swiss National Science Foundation.
 

ACTIONS OF PYRETHROID INSECTICIDES ON INDIVIDUAL SODIUM CHANNEL ISOFORMS

Soderlund, DM. Department of Entomology, NYSAES, Cornell University, Geneva, NY, USA.

Voltage-sensitive sodium channels of nerve, skeletal muscle, and cardiac muscle are important target sites for numerous naturally-occuring neurotoxicants, synthetic insecticides, and drugs. Voltage-sensitive sodium channels in mammals are now known to be the products of a multi-gene family which exhibit both anatomical and developmental regulation of expression. Recently, the molecular cloning and functional heterologous expression of individual sodium channel isoforms has facilitated the description of the pharmacological properties of individual isoforms. Our research focuses on studies of the sensitivity of individual sodium channel isoforms expressed in Xenopus oocytes to pyrethroid insecticides and the correlation of these effects with the diverse actions of pyrethroids on sodium channel function in intact tissues and on the signs of poisoning observed in animals. Research to date has shown that the rat brain IIa isoform, which is expressed abundantly in the adult CNS, is very insensitive to the most potent pyrethroids and completely insensitive to other neurotoxic pyrethroids. These findings imply that one or more isoforms other than brain IIa that are expressed in the CNS are likely to mediate the significant central actions of pyrethroids that are observed following intracerebral administration at low doses. In contrast to these results, a tetrodotoxin-resistant isoform expressed in the rat peripheral nervous system exhibits a sensitivity to pyrethroids that is comparable to the sensitivity of insect sodium channels expressed in this system. The high sensitivity of this isoform may be a significant determinant of the paresthesia caused by some pyrethroids following dermal exposure. Site-directed mutagenesis on this isoform has identified amino acid substitutions that either enhance or reduce the sensitivity of expressed channels to pyrethroids. Our results illustrate the value of this experimental approach in identifying the sodium channels that represent the most sensitive targets for pyrethroids in mammals and the structural domains that serve as determinants of pyrethroid sensitivity.
 

ELECTROPHYSIOLOGICAL TECHNIQUES AND ORGANOPHOSPHATE MECHANISMS

Williams F.M., Toxicology Unit, Department of Environmental and Occupational Medicine, The Medical School, University of Newcastle upon Tyne NE2 4HH, UK

Electrophysiological measurements of peripheral nerve function have particular application to an understanding of the mechanisms of the neurotoxic effects of organophosphates. Most organophosphates are anticholinesterases which produce acute toxicity mainly by peripheral muscarinic effects due to excess acetylcholine, whereas some inhibit neuropathy target esterase (NTE) inducing a delayed peripheral neuropathy (OPIDN). Subtle subcellular changes in the function of the neuromuscular junction and nerve transmission may be detected by measuring jitter; the variability in the latency of action potentials. Jitter measured by single fibre electromyography has been found to be altered in patients with compromised neuromuscular function and in volunteers dosed with sarin.
A model using the isolated phrenic nerve/diaphragm preparation from the mouse has been used to investigate the long term effects of organophosphates on jitter and on neuromuscular transmission following single and multiple doses The acute effects of acetylcholinesterase inhibition at the neuromuscular junction can be detected by measurement of spontaneous minature end plate potentials (meps) the time course of which in believed to relate to the duration of action of acetylchloline. are independent measures of nerve transmission. Studies have been conducted with a range of organophosphate pesticides given as single or chronic doses. Action potential jitter and end plate potential jitter were differentially affected in animals who did not exhibit signs of delayed neuropathy at 7 to 28 days after single and chronic doses at times when enzymes had returned to normal.
Electrophysiological techniques allow investigations of neurotoxicity in model systems that can be directly extrapolated to man

Kelly, S.S., Mutch, E., Williams, Faith M. and Blain, P.G. (1994). Arch. Toxicol. 68 459-466
Kelly, SS, De Blaquiére, GE, Williams, FM. and Blain, PG (1997). Human & Exper. Toxicol. 16 72-78.
 

AGE-RELATED SENSITIVITY TO ANTICHOLINESTERASE PESTICIDES: NEUROBEHAVIORAL AND NEUROCHEMICAL OUTCOMES

Moser, VC and Padilla, S, Neurotoxicology Division, NHEERL, US EPA, Research Triangle Park, NC, USA

Young organisms are unique in many ways, including pronounced differences in physiological and behavioral parameters. The immature nervous system of the young may respond differently to chemical exposure, and/or immature kinetic processes (absorption, distribution, metabolism, excretion) may act on the chemical differently than in the adult. Lately there has been interest, both scientific and regulatory, in the neurotoxic effects of pesticides in the young. The exposure potential of children to anticholinesterase pesticides is higher than most, and the focus of our research has been the carbamate and organophosphorus pesticides. We have shown that the magnitude of age-related sensitivity between young and adult rats depends on the pesticide in question. Maximally-tolerated doses of chlorpyrifos and diazinon are 7-10 times lower in the preweanling rat, whereas there are no age differences with methamidophos. The profile of neurobehavioral responses and neurochemical alterations following pesticide exposure also depends critically on the pesticide. While younger rats are more sensitive to the cholinesterase-inhibiting properties of chlorpyrifos, some behavioral changes are less striking in the young as compared to the adult. In contrast, the magnitude of behavioral responses following methamidophos show no age differences. Age-related differences in sensitivity to some pesticides are probably due, in part, to the lower capacity of the young to detoxify the pesticides. Critical mechanisms for this detoxification include binding to carboxylesterases and hydrolysis by A-esterases. Differences in the neurobehavioral outcomes may be due, in part, to the altered interactions of some pesticides with other neurochemical pathways (e.g., receptor binding), which show different levels of immaturity in the young. We are using both in vivo and in vitro methods to expand our age comparisons of anticholinesterase pesticides.
 

AGE-RELATED DIFFERENCES IN SENSITIVITY TO CHLORPYRIFOS ARE MARKEDLY INFLUENCED BY MAGNITUDE AND FREQUENCY OF EXPOSURE.

Pope, C, Liu, J, Olivier, K, Won, Y and Zheng, Q. Division of Toxicology, Northeast Louisiana University, Monroe, Louisiana, USA.

Unless sufficient data excludes higher sensitivity in children to a particular pesticide, risk assessment under the U.S. Food Quality Protection Act of 1996 now requires incorporation of an additional safety factor for protection of this subpopulation. Several experimental studies have reported higher sensitivity in young animals to organophosphorus (OP) pesticides which act by inhibiting acetylcholinesterase (AChE), but sensitivity has generally been based on lethality following acute exposures. We report here the results of several studies evaluating the relative sensitivity of neonatal (7 days old) and adult (90 days old) Sprague Dawley rats to the OP pesticide chlorpyrifos (CPF) under different exposure conditions. Neonatal rats were more sensitive than adults to acute toxicity following acute subcutaneous (maximum tolerated dosages: 44 vs 279 mg/kg) and oral (LD₁₀s: 15 vs 136 mg/kg) exposures. When both age groups were treated for 14 consecutive days (0, 5 or 10 mg/kg/day, sc), relatively similar neurochemical changes (AChE inhibition and muscarinic receptor binding reduction) were noted in both age groups during dosing (days 8 and 15) but more extensive reductions were observed in adults after dosing (day 22). Relatively similar changes in brain AChE activity and muscarinic and nicotinic receptor binding were also noted in both age groups after 14 daily oral doses of CPF (0, 0.15, 0.45, 0.75, 1.5, 4.5 or 7.5 mg/kg/day). With repeated, intermittent (40 mg/kg, sc every four days X 4) dosing however, more extensive reductions in brain AChE and muscarinic receptor binding were observed in adults. We conclude that age-related differences in sensitivity to CPF can vary qualitatively under different conditions of exposure.
 

GENETIC DIFFERENCES IN SENSITIVITY TO PHARMACOLOGICAL AGENTS

Ellenbroek, BA; Cools AR. Dept. Psychoneuropharmacology, Univ. of Nijmegen, The Netherlands

There is an increasing amount of data that animals differ in their sensitivity to both environmental and pharmacological manipulation. We have found that, when challenged with the dopaminergic agonist apomorphine, Wistar rats show a bimodal type of response. Thus approximately 45% of rats treated with 1.5 mg/kg s.c. Showed less than 10 gnawing counts, whereas another 40% scored more than 500 counts. In order to study the contribution of genetic and early environmental factors in determining the individual susceptibility to apomorphine, we selectively bred rats with a strong response (termed apo-sus) and rats with a weak response (termed apo-unsus). Already after a few generations, there was a strong separation of gnawing response between the two lines, which could be replicated in an independent group of Wistar rats. Cross breeding apo-sus males with apo-unus females and vice versa further supported a role of genetic factors. The offspring of both crosses had an apomorphine susceptibility in between the original selection lines.
In a separate set of experiments, we selectively manipulated the early environment. The results showed that cross fostering apo-sus pup with apo-unsus mothers reduced the apomorphine susceptibility in the pupp, whereas cross-fostering apo-unsus pup with apo-sus mothers had no effect. On the other hand a single 24 hr period of maternal deprivation at postnatal day 9 increased the apomorphine susceptibility in the apo-unsus offspring, but had no effect in apo-sus.
Overall these data show that the susceptibility to apomorphine is determined by both genetic and early environmental factors. Since apo-sus and apo-unsus also differ in a large number of other aspects (including endocrinological and immunological parameters), they offer a valuable model for studying gene-environmental interactions.
 

MULTIPLE CHEMICAL SENSITIVITY (MCS): DIFFERENTIAL DIAGNOSIS IN CLINICAI NEUROTOXICITY.

H. Altenkirch, Department of Neurology, Spandau Hospital, Humboldt University, Berlin.

Multiple Chemical Sensitivity (MCS) is a new constellation of environmental symptoms that has been extensively described and commented on in the USA in the last one and a half decades. The main features of lids phenomenon are multiple symptoms occurring in various body organ systems triggered by a variety of chemical substances with different modes of action; these symptoms recur and exacerbate in certain trigger situations at exposure levels too low to lead to any reactions in the population at large. More than 500 publications on MCS are available and a number of congresses have taken place in the USA. So far it is not clear whether this phenomenon comprises a separate disease entity. In 1996 a WHO symposium on MCS problems reached the conclusion that MCS is not a separate, clinically defined disease. It recommended subsuming it under the more comprehensive term Idiopathic Environmental Intolerance. (IEI) and advised a thorough differential diagnosis of all somatic and psychiatric diseases. This paper tries to describe the neurological and neurotoxic aspects of MCS problems and to illustrate it with examples of an alleged outbreak of chronic neurotoxic disease caused by pyrethroids in Germany.
 

INTERMEDIATE MYASTHENIA SYNDROME FOLLOWING ACUTE ORGANOPHOSPHATES POISONING

Fengsheng He, Fukuang Qin*, Dongren Yang, Haibing Xu, Jingxiang Huang, Institute of Occupational Medicine, Chinese Academy of Preventive Medicine, 29 Nan Wei Road, Beijing 100050, China; *Tengzhou Central People's Hospital, Tengzhou, Shandong, China.

Fifty nine cases out of 687 patients of acute organophosphates (OPs) poisoning were diagnosed as intermediate syndrome (IMS) with a prevalence at 8.58 %. The responsible OPs included parathion, omethoate,dimethoate, dichlorovos and some OP containing pesticide mixtures. Myasthenia was the characteristic clinical feature of IMS which occurred mainly in severe acute OPs poisoning patients after being treated by atropnization and recovered from acute cholinergic crisis at 7-144 hours (the majority being 1- 4 days) from the onset of acute OPs poisoning. Muscular weakness appeared in the following three categories of muscles : (1) neck flexors and proximal limb muscles (100 %); (2) motor cranial nerve innervating muscles (78%); and /or (3) respiratory muscles (67.8 %). Blood acetylcholinesterase activity was persistently inhibited in all cases. Electroneuromyography (ENMG) with repetitive nerve stimulation (RNS) showed decrement of common muscle action potentials at frequencies of 20 Hz or 30 Hz in 5 out of 7 detected patients during the presence of myasthenia. The RNS/EMNG results became normal when the muscle strength recovered. The specific binding of nicotinic receptor of acetylcholine (nAChR) in the lymphocyte membrane was found to be increased in 18 IMS patients compared to 9 controls.
There were 16 mild IMS patients who had myasthenia of the muscles of categories (1) and (2) without the involvement of cranial nerves IX and X innervating muscles. They recovered within 2-7 days with favourite prognosis. Forty three severe IMS patients all needed immediate endotracheal intubation and mechanical ventilation because of respiratory paralysis or weakness of muscles innervated by cranial nerves IX and X producing distress of breathing. The recovery of myasthenia of respiratory muscles and proximal limb muscles was slower than the recovery of the weakness of cranial nerve innervated muscles, the latest being 30 days. Four of them died of respiratory paralysis or multi-organ dysfuntion.
The mechanism of IMS remains to be further investigated. The RNS changes indicate a post-synaptic blockade at the neuromuscular junctions. In order to promote the recognition of this myasthenia syndrome following acute OPs poisoning, we proposed to name the syndrome as "Intermediate Myasthenia Syndrome (IMS)".
 

DRUGS OF ABUSE

J. Henry, Accident and Emergency Dept., St. Mary’s Hostpital, Praed Street, London, W2 1NY, U.K.

Drug addiction is an increasingly important cause of morbidity and mortality in most countries. Diamorphine (heroin) is a common cause of drug related deaths in Britain, but has been superseded by methadone, the drug used in treatment. A major aspect of the toxicity of these drugs is the phenomenon of tolerance. A naïve user can collapse after injection of a few milligrams of heroin while tolerant users average 750 mg, a day without apparent ill effects. However, once use is discontinued, tolerance is lost rapidly and the former regular dose can prove lethal. The stimulant group of drugs, cocaine and the amphetamines (including ecstasy) can cause cardiac arrhytmias, cerebrovascular accidents and hyperthermic collapse. Myocardial infarction has recently been shown to be 23.7 times more likely to occur in the 60 minutes after cocaine use. The ecstasy group of drugs are toxic to serotonin terminals in every animal species studied, but how this relates to human neurotoxicity and human morbidity remains uncertain. Ketamine is neurotoxic in animals, and leaves users feeling "washed out", but the human neurotoxicity is not known. The addictive process itself could be described as neurotoxicity without any objective evidence of damage to nerve cells. Behavioural toxicity deserves attention. Changes in function of discrete neural pathways may ultimately be responsible for the loss of behavioural control which occurs during the natural history of the addition process. Is has been suggested that a complex neural network within the forebrain limbic system mediates drug seeking behaviour and maintains drug self administration in rodents; the same may apply to man.
 

SOLVENTS AND TOXIC ENCEPHALOPATHY – DIAGNOSTIC CHALLENGES IN CLINICAL NEUROTOXICOLOGY

Müller, KMI, Akila R, Päällysaho J, Sainio M. Section of Clinical Neurosciences and BrainWork Laboratory, Finnish Institute of Occupational Health (FIOH), Helsinki, Finland

Annually 80-90 workers are studied at FIOH because of occupational exposure to mixtures of organic solvents and neuropsychiatric symptoms suggestive of organic brain disease. Subjects complain of e.g. memory problems and often present with a clinical picture of "depression". In clinical work the diagnosis of toxic encephalopathy faces many challenges: 1) Quantitative estimation of the level of exposure of an individual to neurotoxic solvents is not accurate. 2) Most subjects are exposed to variable mixtures of industrial organic solvents, and in some cases the additive/modifying effects of alcohol cannot be excluded. 3) The vulnerability of the nervous system to the effects of solvents may differ between individuals. Thus exposure estimated as low level can be significant in some cases but not in others. 4) There are no neurological findings specific for toxic encephalopathy which presents also a challenge for differential diagnostics. 5) A multifactorial aetiology for brain dysfunction is possible. At FIOH the diagnosis of toxic encephalopathy in exposed subjects is based on obtaining evidence of organic brain disease from neuropsychological testing, high field MRI, neurophysiology, vision physiology as well as CSF examinations and a thorough differential diagnostic evaluation for other possible cause(s) of brain dysfunction. All subjects with signs of encephalopathy are followed at FIOH (often for several years) and retested before the final diagnosis of toxic encephalopathy is made (annually 15-20 new diagnoses). With this clinical approach we have gradually obtained a group of patients in whom cognitive performance is currently being studied in more detail to further characterize the nature of their cognitive performance difficulties: In 14 male subjects (44-55 yrs of age and 14 to 37 yrs of exposure to organic solvent mixtures) a cognitive performance profile indicating frontostriatal dysfunction was detected. New challenges are found in the field of neuropsychopharmacological treatment of patients, neuroprotection and early detection of CNS involvement.
 

EXCITOTOXICITY IN THE COCHLEA

Puel JL, INSERM U254 and University Montpellier I - Medical School, Hôpital St. Charles, 34295 Montpellier cedex 5, France.

The use of glutamate as a neurotransmitter of inner hair cell (IHC)-auditory nerve synapse has been firmly suggested in the mid-eighties by anatomo-chemical data, and later on, by physiological experiments using agonists and antagonists. A molecular biology approach has recently confirmed and extended these findings, pointing out to the types and subtypes of glutamate receptors involved. Because of its glutamatergic nature, the IHC-auditory nerve synapse is very susceptible to excitotoxicity: in a first and acute step, the postsynapse is disrupted by an excess release of glutamate linked to anoxia or intense (noisy) stimulation; then, after repetitive attacks, the type-I spiral ganglion neurons may die via a Ca²⁺ induced mechanism. Our previous findings suggest that an excitotoxic disruption of the IHC-auditory nerve synapses, temporarily killing the cochlear potentials, can be followed within 2 to 5 days by synaptic regeneration and a functional recovery. It is essential to understand the molecular mechanisms responsible for this synaptic repair if new therapeutic strategies are to be developed. Experiments, using direct in vivo perfusions with osmotic mini-pumps are in progress to investigate, among several factors, the role of some glutamate receptor subunits (NMDA, metabotropic, etc.) which are well known, in the brain, to play a trophic role during developmental and/or post-traumatic synaptogenesis. First results strongly suggest a trophic function for NMDA receptors in the regrowth of auditory dendrites, the neo-formation of IHC synapses, and thereby the functional recovery. Using a similar protocol, other drugs (such as nerve growth or guidance factors, Ca²⁺ regulators, antibodies or antisenses, etc.) may also be applied.
These experimental findings bode well for the development in the near future of local pharmacological therapies, in human cochlear pathologies where glutamatergic synapses are likely to be involved: i.e., noise trauma and ischemia-related sudden deafness, neural presbycusis and some forms of peripheral tinnitus.
 

ENVIRONMENTAL CONTAMINANTS AND COCHEAR DAMAGE

Fechter, LD, University of Oklahoma Health Sciences Center, Oklahoma City, USA

Cochlear impairment can result from exposure to a variety of drugs (e.g. aminoglycoside antibiotics, loop diuretics, anti-cancer agents), environmental toxicants (e.g. chemical asphyxiants, organic solvents, organic metals), as well as by overstimulation by noise. This presentation will focus on both the exposure conditions and the mechanisms by which chemical asphyxiant exposure can potentiate noise-induced hearing loss. Characterization of risk of such an interaction will be described through dose-response studies. The role of reactive oxygen species in cochlear impairment and the locus of injury will be identified (supported in part by NIOSH grant OH03481).
 

N-METHYLATION AND S-OXIDATION IN HUMAN AND RAT BRAIN

D.B Ramsden*, R.B. Parsons*, J.E. Pritchard^@, M-L Smith,. R.H. Waring and A.C. Williams^@, Departments of Medicine*, Clinical Neurology ^@ and Biochemistry, University of Birmingham, Queen Elizabeth Hospital, Birmingham B15 2TH, UK..

Parkinson’s disease is a consequence of the degeneration of the pigmented dopaminergic neurones of the substantia nigra. Inhibition of complex 1 and oxidative stress are prominent biochemical features. The cause is unknown but both genetic and environmental factors have been implicated. We have explored the molecular biology of pathways that might give rise to the formation of selective nigrostriatal toxins - N-Methylation of pyridines and S-oxidation of cysteine. The former was selected because, following injection of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, the compound is oxidised to the methylpyridinium ion in glial cells, which is taken up by the dopamine re-uptake system of the nigrostriatal neurones where it exerts its toxic action via inhibition of complex 1. N-methylation represents an alternative route to N-methylpyridinium ions. S-Oxidation of cysteine by cysteine dioxygenase regulates intracellular cysteine levels. Excess intracellular cysteine has the potential to react with dopamine to form a series of complex 1 inhibitors.
For both pathways we have adopted a staged approached in which we elucidated the molecular biology of important enzymes: for pathway 1 - nicotinamide-N-methyltransferase (NNMT); pathway 2 - cysteine dioxygenase (CDO) . This involved elucidation of the structures of the mRNA, the gene and its 5'-flanking region. Knowing these we have gone on to explore expression within the brain and factors involved in regulation of the gene.
The 5'-flanking region of the NNMT gene was shown to contain classic glucocorticoid response elements, and cell culture studies indicated that dexamethasone regulated mRNA and NNMT protein synthesis. High N-methylation capacity in humans was shown to be due to synthesis of high levels of NNMT protein and not to any polymorphism with the coding regions of the gene. Western blotting identified regions of high and low expression in human brain and immunohistology shows that the protein is expressed in neurones. .
The structure of human CDO mRNA (1551 b), gene (5 exons) and 5'-flanking region together with chromosomal localisation (5q22-23)of the gene have been elucidated. Enzyme assay and Western blotting have shown the rat basal ganglia to be a site of high, inducible CDO activity. Immunohistology has shown that the protein is expressed in neurones.
Conclusions: Products of both the NNMT and CDO are expressed in CNS neurones. Both genes are inducible. Both are expressed in the basal ganglia. Therefore, both products would appear to be candidates to allow the formation of selective neurotoxins. Further studies are in progress to compare expression in parkinsonian with that of normal brains.
 

PESTICIDE EXPOSURE AND THE PREDISPOSITION TO PARKINSON’S DISEASE

Gorell JM. ^*, Johnson CC ^** , Rybicki BA ^** , Peterson EL ^**, Richardson RJ ^***, Departments of Neurology ^* and Biostatistics & Research Epidemiology ^** , Henry Ford Health System, Detroit, MI; Toxicology Program, Department of Environmental & Industrial Health, School of Public Health, University of Michigan, Ann Arbor, MI ^*** .

We conducted a population-based case-control study with a focus on pesticide exposure and the risk of Parkinson’s disease (PD) in men and women > 50 years of age who were receiving primary medical care at Henry Ford Health System in metropolitan Detroit. PD cases (n = 144) and concurrently recruited, frequency-matched (for age ± 5 years, race and sex) controls (n = 464) were given a questionnaire inquiring about exposure throughout life to herbicides, insecticides, fungicides at work, on a farm, or while gardening.
After adjustment for age, race, sex and smoking status, there was a significant association of exposure to herbicides (OR 4.10; 95%CI 1.37,12.24) and insecticides (OR 3.55; 95% CI 1.75,7.18) and PD, though only in an occupational setting, but no relationship between fungicide exposure and the disease. An association of farming as an occupation with PD (OR 2.79; 95% CI 1.03, 7.55) was maintained after adjustment for occupational herbicide exposure, and was of borderline significance after adjustment for occupational insecticide exposure. No relationship with PD was found with rural living or well water consumption.
These results suggest that PD is associated with occupational exposure to herbicides and insecticides and to farming, and that the risk of farming cannot be accounted for by pesticide exposure alone. These findings will be discussed in the context of other studies of pesticide exposure and PD. New investigative approaches to these issues will also be considered.
 

LEAD: FROM NMDA-RECEPTOR TO LTP.

Wiegand., H; Medical Institute of Environmental Hygiene at the Heinrich-Heine-University, Dusseldorf, Germany

Children exposed to lead (Pb) during development suffer from a range of neurobehavioral deficits including learning and memory disabilities. The developing central nervous system (CNS) has been shown to he highly susceptible to the neurotoxic actions of Pb. Long term potentiation (LTP), a form of synaptic plasticity is assumed to be an appropriate experimental model for learning and memory. This phenomenon of enduring enhancement of increased synaptic activity was recently shown by several investigators to be reduced by Pb. This was true in acute exposure experiments using hippocampal slices in vitro as well as in-vivo treatment. Ex-vivo experiments in hippocampal slices after low level in-vivo treatment of Pb also showed a decreased LTP. Cross fostering experiments revealed a high LTP susceptibility if the treatment included the perinatal CNS development period. The N-methyl-D-aspartate (NMDA) type of glutamate receptor is essential for some forms of synaptic plasticity especially in the hippocampus. The function of this ligand operated ion channel was inhibited by Pb when the animals were exposed both in-vitro and in-vivo. It is possible that the Pb may be interacting at many different sites on the receptor protein thus modulating the receptor function. Besides this type of postranslational Pb interaction with the receptor, there are now several reports to show that Pb can change the receptor protein subunit expression during developmental phase.
The current knowledge reveals a dynamic relationship of NMDA receptor expression and LTP during the developmental neurotoxicity of Pb.
 

Oral Communications:

TRANSFECTION AND OVEREXPRESSION OF METALLOTHIONEIN-I (MT-I) IN MT-I/MT-II KNOCKOUT (MT-KO) MICE INCREASES THEIR RESISTANCE TO METHYLMERCURY (MeHg)-INDUCED CYTOTOXICITY

Aschner, M., Yao, CP, and Allen, JW. Department of Physiology and Pharmacology, Wake Forest University School of Medicine, Winston-Salem, NC, USA

Metallothionein-I (MT-I) protein levels and the ability of MT-I over-expression to protect against methylmercury (MeHg) toxicity were monitored in primary astrocytes isolated from MT-I/MT-II knockout (MT-KO) mice with or without MT-I cDNA transfection, and astrocytes isolated from wild-type (WT) mice. MT-I was expressed by pGFAP-MT-I plasmid transfection under the control of the astrocyte-specific glial fibrillary acidic protein (GFAP) promoter. The levels of MT proteins in astrocytes derived from WT, MT-I/MT-II knockout (MT-KO; MT^-), and MT-I cDNA transfected MT-KO (MT⁺) mice were measured by immunoprecipitation. No MT-I protein was detected in MT-KO astrocytes. MT-I cDNA-transfected MT-KO (MT⁺) astrocytes had significantly (p<0.05) higher MT-I protein levels, compared to astrocytes isolated from either WT or MT-KO mice. The ability of cells over-expressing MT-I to withstand acute methylmercury (MeHg) treatment was measured by the release of preloaded Na₂⁵¹CrO₄, an indicator of membrane integrity. Increased expression of MT-I was associated with attenuated release of Na₂⁵¹CrO₄ upon 5 µM MeHg treatment. In response to MeHg treatment, ⁵¹Cr release was significantly (p<0.05) higher in the MT-KO (MT^-) astrocytes, and this effect was significantly (p<0.05) attenuated in MT-I cDNA transfected MT-KO (MT⁺) cells. ⁵¹Cr release in cells overexpressing MT-I (MT⁺) was also significantly (p<0.05) reduced compared to astrocytes isolated from the WT mouse. These results demonstrate that MT-I can be highly expressed in primary astrocyte cultures by pGFAP-MT-I plasmid transfection. Furthermore, they lend credence to the hypothesis that increased expression of MT-I affords protection against the cytotoxic effects of MeHg, and that MTs offer effective cellular adaptation to MeHg cytotoxicity. Supported by PHS grant NIEHS 07331.
 

ALUMINUNUM SALTS STABILIZE FERROUS IRON AND ENHANCE AGGREGATION OF A TOXIC BETA-AMYLOID FRAGMENT

Bondy, SC; Yang, E; Guo-Ross, SX; Truong, A; Center for Occupational and Environmental Health, Department of Community & Environmental Medicine, University of California, Irvine, USA.

Aluminum is a recognized neurotoxicant in dialysis encephalopathy and may also be implicated in the etiology of neurodegenerative disease, particularly Alzheimer's disease. Alzheimer's disease is suspected to be associated with oxidative stress, possibly due to the pro-oxidant properties of beta-amyloid present in the senile plaques. The underlying mechanism by which this occurs is not well understood although interactions between amyloid, aluminum and iron have been proposed. This study provides a possible explanation whereby both aluminum and b-amyloid can potentiate free radical formation by stabilizing iron in its more damaging ferrous (Fe2+) form which can promote the Fenton reaction. The velocity, at which Fe2+ is spontaneously oxidized to Fe3+ was significantly slowed in the presence of aluminum salts. A parallel effect of prolongation of the stability of soluble ferrous ion was found in the presence of b-amyloid fragment (25-35).
An interaction between aluminum salts and b-amyloid was also looked for. The formation of the beta-pleated configuration of the toxic amyloid peptide fragment, was studied using thioflavin-T fluorescence as an indicator of such folding. Aluminum enhanced the formation of aggregated beta-sheets in a dose dependent manner. This occurred in the presence of phosphate but not tris buffer. It is proposed that a particulate aluminum phosphate complex may form critical nuclei upon whose surface the amyloid peptide can change its configuration. This seeding may be a relevant factor in the formation of insoluble proteinaceous materials such as amyloid plaques which can form a nucleus for initiation of iron-based pro-oxidant events.

Supported by NIH grants AG16794 and ES7992.
 

INHIBITION OF AXON OUTGROWTH IN MOUSE N2a NEUROBLASTOMA CELLS BY TRIORTHOCRESYL PHOSPHATE

Fowler, MJ*, Flaskos, J**, McLean, WG*** and Hargreaves, AJ*. * Department of Life Sciences, Nottingham Trent University, Nottingham, UK; **Faculty of Veterinary Medicine, Aristotelian University of Thessaloniki, Thessaloniki, Greece; ***Department of Pharmacology and Therapeutics, University of Liverpool, Liverpool, UK.

The aim of this work is to develop a cellular system for studying the molecular basis of the neurodegenerative effects of organophosphates. In previous work it was found that subcytotoxic concentrations of tricresyl phosphate (mixed isomers) inhibited axon outgrowth by differentiated N2a cells (Flaskos et al., 1998). In the present study the effects of ortho and para isomers were evaluated, as in vivo studies indicate that these isomers are neuropathic and non-neuropathic, respectively.
In dose response experiments, both isomers inhibited axon outgrowth after 24 hours exposure with IC50 values of 0.7 and 0.9 m g/ml, respectively. However, the effects of TPCP were transient, with no inhibition being observed after 48 hours exposure. Toxicity of TOCP but not TPCP was increased when cells were incubated in the presence of NADPH activated microsomes, indicating that bio-activation into a more toxic metabolite had occurred in the case of the former. In all cases the extent of axon outgrowth correlated well with the levels of neurofilament heavy chain detected on Western blots of control and organophosphate-treated cell extracts. Current work indicates that only the ortho isomer inhibits axon outgrowth following very short exposure times.
This pattern of toxicity correlates well with the toxicity observed with these isomers in vivo. The results are therefore consistent with the view that differentiating N2a cells represent a useful cellular system for studying the molecular events that underlie the delayed neurodegenerative action of organophosphates.

1. Flaskos J; McLean WG; Fowler MJ; Hargreaves AJ (1998) Neurosci. Lett. 242, 101-104
 

INTERFERENCE OF THE ZINC ION IN THE FORMATION OF PYRROLE DERIVATIVES USED AS INDICATORS OF 2,5-HEXANEDIONE NEUROTOXICITY

Mateus, M.L., dos Santos, A.P.M. and Batoréu, M.C., Faculty of Pharmacy of Lisbon, Lisbon. Portugal.

The protective role of zinc against the neurotoxic effects of 2,5-hexanedione (2,5-HD), was investigated by subacute in vivo experiments and the molecular mechanism of this interaction was performed using in vitro systems. For one week male Wistar rats were exposed i.p. (3 groups) to 2,5-HD, 2,5-HD plus zinc acetate and zinc acetate. Rats not exposed were used as control. During the treatment period, pyrroles were determined in urine, using Ehrlich´s reagent. The molecular mechanism of this interaction was investigated incubating 2,5-HD (240mM) with N-acetyllysine (24mM) in borate buffer following addition of zinc acetate(12mM) under nitrogen atmosphere at 37ºC, in the dark, and the same experience was performed without zinc acetate. Pyrrole reaction products were detected with Ehrlich´s reagent and separated and characterized by HPLC and GC/MS. The in vivo experiments show a significant decrease of excretion of pyrroles in rats co-exposed to the mixture(2,5-HD+zinc) when compared with the values obtained with rats exposed to 2,5-hexanedione alone. In the preliminary results of in vitro assays it was observed a decrease in the determination of pyrroles when 2,5-HD is incubated with N-acetyllysine in the presence of zinc acetate when compared with the pyrroles formed in the absence of zinc. Therefore,in both experiments the results confirm the protective role of zinc in 2,5-HD neuroxicity. The mechanism of this interaction is now under study.
 

STYRENE-INDUCED CHANGES IN DOPAMINERGIC RETINAL CELLS. AN EXPERIMENTAL STUDY IN THE RAT.

M.V. Vettori., D. Corradi*, T. Coccini^§, S. Cavazzini, A. Carta°, L. Manzo^§, and A. Mutti. Department of Clinical Medicine, Nephrology & Health Sciences; *Institute of Anatomy Pathology, °Institute of Ophthalmology, University of Parma Medical School, ^§ Maugeri Foundation Medical Centre, University of Pavia, Italy.

Background: Dopamine (DA) is synthesised in amacrine cells and released upon membrane depolarisation in a calcium-dependent way. Thus, it is recognised to function as a major neurotransmitter or modulator in vertebrate retina. Owing to DA modulating activity on cone-horizontal cells transmission, depletion or dysfunction of amacrine cells could interfere with chromatic processing, accounting for the acquired dyschromatopsia described among styrene-exposed workers.
Aim of the study: The present study has been designed to test the hypothesis that amacrine cells represent a selectively vulnerable target of styrene in subchronically exposed rats.
Experimental design and methods: Ten female Sprague-Dawley rats were exposed to 300 ppm styrene 6 h/day, 5 days/week, for 12 weeks; ten pair-fed rats exposed to fresh air served as a control group. Whole mounted retinas were used for the morphometry of TH reactive cells. DA content and tyrosine hydroxylase (TH) activity were measured by HPLC and electrochemical detection.
Results: In treated rats, morphometric analysis showed a loss of TH immunoreactive (IR) cells (6.1/mm² vs. 9.0/mm² recorded in controls, t = 4.6, p < 0.001), without any peripheral-central variation in cell loss. DA content was also lower in exposed, as compared to control animals (306.9 vs. 230.2 ?g/g w.w., t = 2.63, p = 0.015).
Conclusions: Retinal TH-IR are sensitive to styrene exposure, which seems to cause both structural and functional changes, represented by cell loss and DA depletion, respectively. Such changes may explain the loss in chromatic discrimination recorded among workers occupationally-exposed to styrene.

Acknowledgements: Supported by European Commission (ENV-CT96-0173).
 

NOCTURNAL OXYGEN SATURATION IN LONG-TERM SOLVENT EXPOSED WORKERS.

M.K. Viaene, G. Laire, G. Dours, B. Nemery. Department of Occupational Health. Catholic University of Leuven, Belgium.

Introduction: Recently, it has been suggested that occupational exposure to solvents may cause an increase of nocturnal sleep apnoeas (Laire et al. 1997).
Methods: A polysomnographic examination (Alice 3) was performed in a group of long-term solvent-exposed printers (n=21) and age matched controls (n=27) during one night at home. In addition, a self-administred standardized questionnaire was completed. The groups were well comparable regarding age (exposed: 34,8 ± 6,5; controls: 32,7 ± 6,5), weight (78,9 kg ± 16,8; 80,1 kg ± 10,1), neck circumference (% predicted for length 97,6% ± 10,3; 96,2% ± 5,7), alcohol use (1,0 ± 1,1 ; 1,7 ± 1,8 beers/day), and smoking (7,1 ± 8,5 ; 11,9 ± 11,1 cigarettes/d). The average exposure duration was 15 years (± 10). Historical biomonitoring and environmental monitoring results were available for all jobtitles (17% to 185% of the cumulative threshold limit value). A semi-quantitative cumulative exposure index (EI) could be calculated. Alcohol use, history of alcohol abuse (years), neck circumference, age and smoking were used as covariables in the multiple regression analysis.
Results: The exposed workers reported an increased sleepiness in the evening (p<0,01) and a decreased sexual functioning (p=0,03). The polysomnography results showed an increased incidence of central apnoeas (1,8 events/hour ± 2,7 vs. 1,4 ± 4,1; p<0,01), increased mean duration of central apnoeas (9,3 seconds ± 7,2 vs. 4,7 ± 8,9; p=0,06) and hypopnoeas (18,1 seconds ± 5,7 vs. 9,7 ± 7,6; p<0,01) in the printers vs. the control workers. Sixty-seven percent of the exposed workers experienced central apnoeas and all experienced hypopnoeas during the night compared to 29,6% and 66,7% of the control workers (FE test, both p<0,01). All these outcome parameters were significantly related to the exposure index (multiple linear or logistic regression, p<0,04). As was reported in the study of Laire et al. 1997, all effects on night-time breathing were almost solely present in the exposed non-smokers (n=10).
Discussion: These data confirm the negative influence of solvent exposure on nightly oxygen saturation. Non-smokers may be especially vulnerable. This might be due to the absence of the central stimulating effect of nicotine.

References: Laire G, Viaene MK, Veulemans H, Masschelein R, Nemery B. Nocturnal oxygen desaturations, as assessed by home oximetry, in long-term exposed works. Am J Ind Med 1997; 32:656-664.
 

Posters: Metals

STUDIES ON PROTECTIVE FACTORS FOR CELL DEATH OF CEREBELLAR NEURONS INDUCED BY METHYLMERCURY

Adachi, T; Ishido, M; Kunimoto, M. Regional Environment Division, National Institute for Environmental Studies, Tsukuba, Ibaraki 305-0053, Japan

It has been well documented that neuronal damage by methylmercury (MeHg) is remarkably selective, being limited to specific focal areas including cerebellar granule cells. We recently found that neuronal cell death was apoptotic using primary cultures of cerebellar neurons, and that caspase inhibitors did not protect the cell death. In the present study, we investigated the protective effect of anti-oxidants on MeHg toxicity to determine whether oxidative stress is involved in the cell death. Primary cultures of cerebellar neurons were prepared from newborn Wistar rats, and maintained in serum-free medium for 2 days before use in the experiments. When MeHg was added to the cultures at 30 nM, cell viability decreased rapidly after 2-days lag-period. Simultaneous addition of alpha-tocopherol, Trolox or probucol (20 microM each) with MeHg inhibited the decrease of cell viability. Sodium selenite (100 nM) also delayed the cell death caused by MeHg. Furthermore, alpha-tocopherol was effective even when it was added 2 days after the addition of MeHg, and the protective effect lasted at least for 3 days. However, these agents did not protect the cell death by higher doses (>300 nM) of MeHg. These results suggest that oxidative stress is involved in apoptotic cell death of cerebellar neurons induced by a low dose of MeHg at a downstream of apoptotic cascade. It is also suggested that alterations during 2-3 days after the addition of MeHg are critical for the neuronal cell death.
 

INVESTIGATION OF COMBINED SUBCHRONIC LEAD, MERCURY AND ETHANOL EXPOSURE ON NEUROPHYSIOLOGICAL PROCESSES IN RATS.

Desi, I., Papp, A., Nagymajtenyi L. Department of Public Health Albert Szent-Gyoergyi University Medical School and WHO Collaborating Centre for Chemical Safety, Szeged, Hungary.

In our previous study it was found that low dose lead and mercury administration altered dose-dependently and significantly some neurophysiological parameters. The aim of this study was to investigate the changes of the neurophysiological processes of rats administered parallel with low doses of lead/mercury and ethanol.
12 weeks old male Wistar rats were treated orally by gavage with 80.0 and 320.0 mg/kg lead (in form of lead-acetate) or 0.4 and 1.6 mg/kg mercury (in form of mercury-chloride) or with ethanol (5%) dissoloved in drinking water, or with the combination of the mentioned doses of the metals and ethanol for 12 weeks. The neurophysiological functions investigated were: ECoG, cortical evoked potentials, conduction velocity, relative and absolute refractory periods of the peripheral nerve.
The body weights were lower in the treated groups, especially in the case of the combinations, but the differences were not significant. Clinical symptomes of chronic lead/mercury or ethanol intoxications were not seen. The electrophysiological parameters were dose- and treatment variation-dependently altered. In case of the higher metal doses and metal+ethanol administration some changes compared to the control were significant, while compared to the alterations to the single ethanol or lead/mercury administration they were not significant but more expressed.
The results showed that the combined ethanol and metal exposure caused more serious functional neurotoxicological effects. Although the experimental data can not be transferred directly to humans, one can suppose that this combination can also be a real risk for the low level exposed human population. The study was supported by the Hungarian ETT grant no T08128.
 

COMPARISION OF THE DETECTION SENSITIVITY OF NEURO- AND IMMUNOTOXICOLOGICAL APPROACHES IN DETECTION OF SUBACUTE INORGANIC MERCURY EXPOSURE IN RATS

Institóris, L, Siroki O, Nagymajtényi L, Papp A, Dési I. Dept. Public Health, Albert Szent-Györgyi Med. Univ., Szeged, Hungary.

Following per os administration of 1.6, 0.8, and 0.4 mg/kg mercury (as HgCl₂) to 4 weeks old male Wistar rats for 28 days, certain toxicological, neurological, and immune function parameters were determined. The investigated parameters were: body weight gain, weights of nine organs, electrocorticogram (ECoG), somatosensory, visual, and auditory evoked potentials, conduction velocity of the tail nerve; as well as PFC content of the spleen and DTH reaction. Among the general toxicological parameters only the relative kidney weight (related to both 100 g body weight and brain weight) increased in a dose dependent manner (p<0.05 at the highest dose). Mean amplitude of the somatosensory, visual and auditory ECoG, and the conduction velocity of the tail nerve, showed a decreasing tendency with increasing dose, while ECoG mean frequency, latency of the evoked potentials and refractory periods of the tail nerve increased with higher doses. All neurophysiological changes were seen as tendencies only; statistically significant differences from the control group were not found. Among the immune functions examined, only the PFC content of the spleen increased significantly at the highest dose (p<0.05).
According to these results, the immune system seems to be more sensitive to the repeated low dose inorganic mercury exposure than the nervous system.

Supported by the Hungarian OTKA grant No. T-026467.
 

Developmental effects of combined lead, mercury and ethanol exposure on neurophysiological processes in rats

Nagymajtényi L., Dési I., Schulz H., Papp A., Vezér T. Department of Public Health, Albert Szent-Györgyi Medical University, Szeged, Hungary

The results of our previous investigations showed that low dose of lead, mercury administered in the different stages of ontogenesis dose and phase-dependently altered certain neurophysiological processes. In this study the alterations of some neurophysiological parameters of rats simultaneously treated by lead/mercury and ethanol were analyzed.
Female Wistar rats (P generation) were treated by gavage with 80.0 and 320.0 mg/kg lead (in form of C₄H₆O₄Pb· 3H₂O), 0.4 and 1.6 mg/kg mercury (in form of HgCl₂), 5 % ethanol in drinking water, or with the combination of the mentioned doses from day 5 to 15 during pregnancy, or from day 5 to 15 of pregnancy + for 4 weeks of lactation, or from day 5 to 15 of pregnancy + for 4 weeks of lactation + the male offspring (F1 generation) for 8 weeks after weaning. The neurophysiological investigations (ECoG, cortical evoked potentials, etc.) were performed at the age of 12 weeks of F1 rats.
The number of newborn rats/litter, their average weight and the body weight gain of the treated rats were lower, but the differences compared to the control were not significant; clinical symptomes of lead, mercury, or ethanol intoxication were not seen even in the groups administered their combination. The electrophysiological parameters were dose-, combination- and treatment variation-dependently changed, in case of the higher lead/mercury dose, and lead/mercury + ethanol administration the alterations compared to the control were significant, compared to the single lead, mercury administration were more, but insignificantly expressed.
The results showed that, depending on the period of administration during the development, the combined lead/mercury and ethanol exposure caused considerable neurophysiological alterations of the central and peripheral nervous system without any other toxicological changes. One can suppose that the combination of these chemicals can also be a real, higher risk for human being, especially during the early development of the nervous system.

The study was supported by the Hungarian ETT grant No T08128.
 

THE INFLUENCE OF COMBINED TOXIC EXPOSURE ON IN VIVO HIPPOCAMPAL POPULATION SPIKES IN RATS

Papp, A. and Nagymajtényi, L. Department of Public Health, Szent-Györgyi Albert Medical University, Szeged, Hungary

Several toxicants of, e.g., food or environmental origin are known to affect the nervous system but our knowledge on effects of combined exposures is limited. The aim of the present work was to study the effects of a combination (subchronic + acute) of some well-known neurotoxic substances on the hippocampal population spike (POPSP) in rats.
Ten weeks old male Wistar rats (ca. 300 g b.w.) were treated with ethanol (5 % in the drinking water) or with lead (320 mg/kg) mercury (1.6 mg/kg) or dimethoate (1/100 LD₅₀) by gavage for 6 weeks (control rats received normal water and no gavage). Then, the animals were anaesthesized with urethane (1000 mg/kg) and the left hemisphere was exposed. A peritoneal cannula was placed for acute administration.
For evoking and recording hippocampal POPSPs, a bipolar stimulating electrode was positioned at the perforant path while the recording glass microelectrode was put in the CA1 region. The perforant path was stimulated by a train of 20 stimuli at 0.1 Hz every 10 minutes. The POPSPs were averaged and latency, duration and amplitude was measured. Individual relative changes and basic statistical parameters (mean and SD for whole groups of 10 animals) were calculated. After taking 5 - 6 control records, lead, mercury or dimethoate was administered acutely ip., and further records were taken for at least 90 min. In the pre-treated groups, the effect of the subchronic treatment on the POPSPs was, compared to untreated controls, clearly seen. It was partly dissimilar to the effect of the same substance (metals and dimethoate) on acute administration, mainly because the effect was seen also on the latency and duration, not only on the amplitude. If the same agent was given again in a 5 to 10 times higher acute dose, the changes of the POPSP showed a mixed feature of the known acute and the subchronic effects. Alcohol pretreatment made the POPSP more difficult to elicit, and the effects of one of the the agents administered acutely were diminished and delayed as compared to non-pretrated animals. Hippocampal population spikes seem thus suitable for investigating combined neurotoxic actions.

Supported by the Hungarian ETT grant N^o T 08128.
 

DOSE-DEPENDENT INCREASE OF EXTRACELLULAR MATRIX PROTEINS IN SKELETAL MUSCLE OF YOUNG RATS AFTER PRE- AND POSTNATAL LOW LEVEL LEAD EXPOSURE

Stoltenburg, G*, Trebing, A*, Wiegand, H**, von Moers, A***. *Dept. of Neuropathology, Free University Berlin, Berlin, Germany;**Institute of Environmental Hygiene, Düsseldorf, Germany, ***Dept. of Pediatrics, Humboldt-University Berlin, Germany

Chronic low level lead exposure results after years of exposure in myopathy with fibrosis (1). To evaluate the earlier stages and to elucidate the development of the fibrosis, different extracellular matrix (ECM) proteins of skeletal muscle of young rats were studied. Female Wistar rats were fed a diet containing 1500 ppm, 750 ppm or 0 ppm lead acetate in the standard food, for 5 months prior to mating and during the whole gestation and suckling period. The offspring of the lead exposed and control dams were investigated at the postnatal age of 17 to 21 days.
Frozen sections of musculus vastus medialis were investigated by immunohistochemistry using antibodies to collagens type I, III, VI, and XIV and to non-fibrillar glycoproteins fibronectin and tenascin. Morphometric analysis of the ECM components showed a dose-dependent increase. Additional ultrastructural investigation revealed a large number of mastcells among fibroblasts with activated endoplasmic reticulum in the lead exposed animals.
The nature of the increase of ECM components after low level lead exposure is either due to increased production or decreased resolution by metalloproteinases. Satellite cells in skeletal muscle are producing metalloproteinases. Their development might be retarded or their action might be disturbed by lead. A primary fibrosis could be the result of affection of calcium homeostasis the disturbance of which may account for progression of fibrosis.
Buchheim K; Stoltenburg-Didinger G; Lilienthal H; Winneke G. (1998) NeuroToxikology 19: 539-546.
 

METHYLMERCURY TOXICITY TO CATECHOLAMINERGIC (PC12) AND GLIAL (C6) CELLS.

M.V. Vettori., R. Alinovi, R. Gatti*, S. Belletti*, G. Orlandini* and A. Mutti, Department of Clinical Medicine, Nephrology & Health Sciences; *Institute of Histology, University of Parma Medical School, Italy.

Treatment of PC12 and C6 cells with MeHgOH (10^-3-10^-8 M) was followed by time- and dose-dependent morphological, biochemical, and functional changes.
Cell necrosis and apoptosis. Dose-dependent cell necrosis was observed in PC12 cells, the response rate varying from 10 to 100 % after 3 h exposure to 10^-7 - 10^-4 M MeHgOH. Changes consistent with apoptosis were never seen in PC12 cells. Similar effects on cell viability with an overlapping dose-response relationship were observed in C6 cells, which however undergo apoptosis, but not necrosis, after exposure to MeHgOH.
Biochemical changes. Both intra- and extra-cellular concentrations of dopamine (DA) and DA metabolites were reduced in a dose-dependent manner proportional to the cell death rate at 3, 6 and 24 h, whereas tyrosine hydroxylase content and activity in surviving cells were not affected by MeHgOH treatment. Neither hydroxy adducts to DNA (8-OHdG) nor intracellular glutathione (GSH) - measured as markers of oxidative stress - were affected by MeHgOH in PC12 cells, whereas such markers showed dose-related changes in C6 cells.
Conclusions. After exposure to MeHgOH, cell necrosis and apoptosis seem to represent the critical effects in PC12 and C6 glial cells, respectively. Whereas the mechanisms involved in MeHgOH-induced necrosis in PC12 cells has yet to be determined, GSH depletion and reduced scavenging capacity seem to be account for the apoptotic response to oxidative stress by C6 cells.

Acknowledgements: Supported by European Commission (ENV-CT96-0173).
 

Posters: In Vitro approaches

NEURONAL EFFECTS OF MICROBIAL TOXINS, LIPOPOLYSACCHARIDE AND FUMONISIN B₁, ARE NOT MEDIATED THROUGH OXIDATIVE STRESS IN HUMAN NEUROBLASTOMA CELLS

Loikkanen J*; Vähälassi L*; Tapaninen R-L*; Savolainen K**. *Department of Pharmacology and Toxicology, University of Kuopio, Finland; **Department of Occupational Hygiene and Toxicology, Finnish Institute of Occupational Health, Helsinki, Finland.

The mold house problem leads to exposure to a large number of microbes and microbial toxins. The typical symptoms of exposure to indoor microbial bioaerosols are fatigue and respiratory tract disorders such as infections, irritation of respiratory airways and lungs, and febrile reactions if exposed to sufficiently high concentrations. Moreover, recent observations indicate that microbial toxins such as lipopolysaccharide (LPS) and fumonisin B₁ (FB₁) may cause serious effects to central nervous system (CNS). We have studied the effects of LPS and FB₁ on the production of reactive oxygen species (ROS), intracellular levels of glutathione (GSH) and cell viability in human SH-SY5Y neuroblastoma cells. The cells were exposed to LPS (0, 0.1, 1 and 10 ?g/ml) and FB₁ (0, 1, 10 and 100 ?M) up to 6 hours in 48-well plates. The production of ROS, the levels of GSH and cell viability was measured at 0, 0.5, 1, 2, 3, 4, 5 and 6 hours by using fluorescent probes, dichlorofluorescein, monochloro-bimane and propidium iodide, respectively. At these time points the exposure to LPS or FB₁ did not have any significant effect on the production of ROS, the levels of intracellular GSH and cell viability compared to control cells. These data suggest that oxidative stress is not involved in the direct acute neuronal effects caused by LPS and FB₁. Therefore, other mechanisms, by which LPS and FB₁ cause CNS effects, should be studied. It is possible that activation of microglia is a major factor at least in mediating LPS-induced CNS effects. Supported by the Academy of Finland.
 

COMPARISON OF IN VITRO NEUROTOXICITY OF ARTEMISININ DERIVATIVES: ROLE OF CELLULAR GLUTATHIONE

McLean, WG; Sadler, CJ; Dodd, CC; Smith, SL; Edwards, G; Ward, SA; Park, BK. Department of Pharmacology, University of Liverpool, Liverpool, UK.

The antimalarial peroxides dihydroartemisinin and artemether display neurotoxicity in animals in vivo and in neuronal cells in vitro, probably due to the production of free radicals. This work compares the mechanisms of toxicity of artemether, which requires haem-bound iron, with the metabolite dihydroartemisinin, which does not.
NB2a neuroblastoma cells were differentiated for 24 h in the presence of the drugs; toxicity was measured as a reduction in neurite outgrowth. Artemether with haemin (0.3 m M/ 2 m M) and dihydroartemisinin (1 m M) reduced neurite outgrowth to 19.6 ± 15.2 % and 11.5 ± 11.0 of control, respectively (both P<0.001, n=4). The effect of artemether/ haemin was completely prevented by the enzymic antioxidants superoxide dismutase and catalase (109.7 ± 14.1 % and 107.0 ± 29.3 % respectively; P<0.001 compared with drug alone, n=4). In contrast, neither enzyme affected dihydroartemisinin toxicity. Glutathione (0.1 mM) completely prevented toxicity of artemether/ haemin (123.8 ± 12.4 %), but only partially reduced toxicity of dihydroartemisinin (57.9 ± 23.4 %; both P<0.001, n=4).
Glutathione (in its reduced form) was measured by HPLC with fluorescence detection. Artemether/ haemin lowered intracellular glutathione to 27.6 ± 18.9 % of controls (P< 0.01, n=4). Dihydroartemisinin did not affect glutathione (96.2 ± 5.9 %, n=4), nor did haemin alone. Glutathione was lowered to 9.5 ± 4.7 % by incubation of cells with buthionine sulphoxamine, with no effect on neurite outgrowth.
We conclude that the mechanisms of toxicity of artemether and dihydroartemisinin differ and that their toxicity is not solely a consequence of a reduction in intracellular glutathione.

Supported by the Wellcome Trust and the Medical Research Council, UK.
 

INSULIN REVERSES THE AMINOGLYCOSIDE-INDUCED REDUCTION IN EXPRESSION OF HAIR CELL-SPECIFIC CYTOSKELETAL PROTEIN mRNA IN SINGLE CHICK UTRICLES IN CULTURE

McLean, WG; Stacey, DJ. Department of Pharmacology, University of Liverpool, Liverpool, UK.

We have previously shown that incubation of isolated chick utricles in culture for 48 h with neomycin leads to selective down-regulation of mRNA for the hair cell-specific proteins fimbrin and c-b 4-tubulin, relative to the non-hair cell-specific protein b -actin. These changes persist for a further 4 days of incubation in the absence of drug. The purpose of this work was to determine factors that might reverse these changes.
Utricles from 1 day-old chicks were cultured individually in serum-containing medium. After 24h, 1 mM neomycin or 1 mM gentamicin were added to the medium, in the presence or absence of insulin (5 m M). Utricles were incubated either for 48h, or for 48 h and then for a further period of 4 days in the absence of aminoglycoside. RNA was extracted from each utricle, reverse transcribed into cDNA and amplified with specific primers. Products were separated on agarose gels and quantified by densitometry.
At 48 h, gentamicin reduced the ratio of fimbrin mRNA to b -actin mRNA from 1.3 to 0.06 in the absence of insulin and 0.2 in the presence of insulin (both P<0.01 by Kruskal-Wallis test with Dunn’s correction, n=8). After a further 4 days, the ratio remained reduced from 2.7 to 0.2 without insulin, but was restored to 2.3 with insulin (P<0.01, n=8). Similar statistically significant results were obtained with neomycin and with mRNA for c-b 4-tubulin. Histology showed hair cell loss after 48h and 4 days of incubation with aminoglycosides. The results with insulin suggest that restoration of hair cell-specific protein mRNA may be an early marker for recovery of structure.

Supported by the Medical Research Council, UK.
 

THE RECOVERY OF KEY ENZYMES IN HEN EMBRYO BRAINS SPHEROIDS FOLLOWING ACUTE EXPOSURE TO ORGANOPHOSPHATES

K.M.Sales^*, K.M.Doyle^*, S.T.Kingston^**, C.KAtterwill^** & W.M.Purcell^***. *University of Hertfordshire, Hatfield, UK, **Roche Discovery Welwyn, UK, ***University of the West of England, Bristol, UK.

The regulatory test assessing the potential of organophosphates (OPs) to induce delayed neuropathy is the hen test. This correlates behaviour in OP treated hens with brain neuropathy target esterase (NTE) levels and histological assessment of neuronal damage. Hen Embryo Brain Spheroids (HEBS) express both NTE and acetylcholine esterase (AChE), key enzymes in the mechanisms of OP induced toxicity. HEBS are potentially useful in vitro models for differentiating the toxic effects of the OPs.
HEBS were cultured for 14 days in neurobasal media + N2 supplement serum free media with 30 nM LT3 and rotated at 75 rpm and 37° C. HEBS were treated with a single concentration of OP for 24 hours and harvested over 21 days. NTE and AChE were assayed at each time point and expressed against total protein. Results are shown as mean (± SEM) of four experiments.
Inhibition of NTE and AChE in HEBS agreed with effects reported in the hen test. Paraoxon initially reduced AChE to 3% of controls (p<0.01) showing recovery to 55% of controls on day 21. Malaoxon inhibited both NTE and AChE showing rapid recovery to control levels. Leptophosoxon inhibited AChE, 6.2% (p<0.01) of controls, recovering to 53.3% of controls on day 21; NTE also recovered from inhibition to 6.3% (p<0.01) of controls to 61.0% on day 21. It is proposed that the HEBS model shows continuing promise as a pre-screen for the delayed neurotoxic effects of OPs.

Funded in part by the Ministry of Agriculture Fisheries and Food
 
 

Posters: Neurobehavioural toxicology

REGULATORY BEHAVIOURAL TESTING OF PRE-WEANLING RAT PUPS

Collier, MJ; Gower, AJ; Hazelden, KP; Myers, DP. Huntingdon Life Sciences, Eye Research Centre, Eye, Suffolk, UK.

As part of regulatory requirements for developmental neurotoxicity studies, the US EPA guidelines require evidence of competence in detection of effects in offspring. This includes examination outside the home cage at 4, 11, 21, 35, 45 and 60 days of age. From 21 days of age, the methodology established for testing adults is considered appropriate. However, for pups aged 4 and 11 days, specific methodology was developed, taking into account their limited repertoire of behaviour. Testing consisted of evaluation of surface righting reflex, immediately followed by observing behaviour in an arena marked into sectors. Locomotor activity (number of sectors entered), distance travelled and the extent of pivoting were assessed for 4-day old pups placed in a circular arena (diameter 18 cm). Locomotor activity, rearing, grooming and urination were assessed for 11-day old pups placed in a rectangular arena (30 x 21cm). Other behaviours were recorded descriptively. Comparison of non-treated Sprague-Dawley and Wistar rats indicated a marked strain difference in baseline values at 4 days of age; reduced extent of pivoting and a lower level of activity was observed in Sprague-Dawley pups. Ability to detect treatment-related changes was assessed in Sprague-Dawley pups by evaluating behaviour 30 minutes after direct gavage administration of d-amphetamine at 2 mg/kg or di-isofluorophosphate (DFP) at 3.0 mg/kg. Surface righting reflex was facilitated by DFP in 4-day old pups, while activity (sectors crossed) was increased by d-amphetamine in 11-day old pups. These investigations demonstrated the ability of this methodology to detect behavioural changes in 4- and 11-day old pups.
 

BEHAVIOURAL TOXICITY OF DOMOIC ACID AND KAINIC ACID IN NEONATAL RATS

Doucette, TA; Strain, SM; Tasker, RAR. Department of Anatomy & Physiology, Atlantic Veterinary College, UPEI, Charlottetown, Prince Edward Island, Canada, C1A 4P3

Domoic acid (DOM) and kainic acid (KA) are both naturally occurring excitotoxins known to exert their main effects by acting as agonists at AMPA/kainate subtypes of glutamate receptors. Our objective was to compare the behavioural toxicity profiles of DOM and KA in newborn rats at various stages of postnatal development. Adult Sprague-Dawley rats (200-300g) were housed in a colony room with food and water available ad libitum. Females were harem-mated with males and the time of birth for each litter was recorded. For each litter the first 24 hours following birth was defined as post-natal day 0 (P0). Groups of rat pups (N=6 for each) were administered saline or varying doses of DOM on P0, 5, 8, 9, 14, 22 and 30 (females) or 50 (males). Dose response curves for KA were constructed on P8 and P14 for both sexes. Toxicity was recorded as both cumulative behavioural toxicity according to a 4 point scale and as latency to the onset of persistent motor seizures. Analysis of the data revealed that toxicity profiles for DOM were similar in animals up to P14 but that a significant decrease in potency occurred between P14 and P22 (approximately 8 fold). In contrast, the potency of KA decreased by approximately 10-fold between P8 and P14. Prior to P14 KA was about one fourth as potent as DOM whereas this difference increased to almost 10-fold at P14. Behavioural differences between animals receiving DOM or KA were also noted. We conclude that a currently unidentified mechanism effectively dissociates between KA and DOM-induced neurotoxicity at certain stages of neonatal development.
 

NEUROTOXIC EFFECTS OF DAILY ORAL EXPOSURE TO LOW LEVELS OF TRIMETHYLTIN (TMT) CHLORIDE OVER A 22 DAY PERIOD

Haggerty, GC; Morton, S; and Levin, S. G.D. Searle & Co., Skokie, IL, U.S.A.

This study demonstrated that subchronic exposure to TMT (an organotin known to cause neurotoxicity after acute exposure) at oral doses as low as 0.4 mg/kg/day for up to 22 days caused neurotoxicity in rats, the severity of which was dependent on dosage, time of death or sacrifice and sex. Groups of Charles River CD rats (15/sex/group) received doses of 0.4, 0.75 and 1.5 mg/kg/day up to 22 days and were behaviorally assessed (using a functional observational battery and an automated test of locomotor activity) on a weekly basis. Deaths occurred in the 0.75 and 1.5 mg/kg/day groups and as early as Day 11. Development of TMT-induced behavioral effects in the mid and high dose groups was observed earlier in males than in females. In the 1.5 mg/kg/day males, increased open field rearing activity (after 2-3 doses) progressed to hyperactivity and aggressive behavior by Day 9 and to convulsions by Day 10; by Day 14, all surviving animals in this group were euthanized due to moribundity. In the 0.75 mg/kg/day group, a similar temporal pattern of behavioral effects was seen as early as Week 2 in the males and Week 3 in the females. No effects on neurobehavioral function were observed in the 0.4 mg/kg/day animals. The most consistent histological finding seen at all dose levels was necrosis of the hippocampal neurons, in particular the CA1, CA2, CA3 and CA4 neurons (severity was higher in males than females). Neuronal necrosis was also noted (0.75 and 1.5 mg/kg/day) in the brain stem, subcortical nuclei and selected areas of the cortex.
 

SCHEDULE-CONTROLLED OPERANT BEHAVIOR AS A MEMORY MEASURE IN RATS

Miyagawa, M; Ohtani, K; Honma, T. National Institute of Industrial Health, Ministry of Labour, Kawasaki, Japan.

A schedule of reinforcement for operant conditioning (alternating mix FR 10 DRO 10 sec with timeout) was developed for assessing the effects of chemicals on short-term memory in rats. The FR component (in which rats had to respond 10 times to obtain a food pellet) and the DRO component (in which food presentation was contingent upon no responding for 10 sec) alternated after each reinforcement in this schedule. A timeout of variable duration (from 4 to 20 sec) was imposed between presentation of the schedule components as a delay interval. No exteroceptive stimuli were available to indicate which component was in effect. Consequently, appropriate performance depended on the rats' memory of the component presented previously, and the accuracy of behavior alternation was used as a memory measure. The effects of drugs on the delay-accuracy curve (retention gradient) were examined with two different types of response (lever-press and nose-poke). After administration of Scopolamine, Mecamylamine and Methamphetamine, response rate and the first response latency in each FR and DRO component were determined. The first response in each component was classified into four categories (hit, miss, false alarm, and correct rejection) by using its latency with a criterion of 10 sec, and then the probability of hit and false alarm were used to calculate the accuracy of response (A=P[Hit]-P[FA]) and response bias (B=P[Hit]+P[FA]-1). All tested drugs decreased overall response accuracy dose dependently. Effects on retention gradient (larger effects with longer delays) were revealed clearly in the case of methamphetamine (lever-press). The alternating mixed-schedule is effective not only as a performance measure but also as a memory measure. Supported in part by the Science and Technology Agency, Japan.
 
 

Posters: Human Toxicology

CLINICAL AND NEUROPHYSIOLOGICAL INVESTIGATIONS IN A GROUP OF PEST CONTROL OPERATORS. A CONTROLLED STUDY.

Altenkirch, H.; C. Schellschmidt; G. Walter; D. Hopmann; B. Brockmeier, Department of Neurology, Spandau Hospital, Humboldt University Berlin

Acute pesticide intoxication by pyrethroids, organophosphates and carbamates are clinically and neurophysiologically well defined entities. Less data exists on the putative chronic neurotoxic effects of pesticides after long term low level exposure.
We performed a controlled study of 21 professional pest control operators (PCOs) exposed to pyrethroids, organophosphates and carbamates and 20 controls matched for age and sex. The study programme consisted of a detailed neurological, medical and occupational history and physical examination, nerve conduction studies, electroencephalography, electrocardiography and laboratory investigations. Mean time of occupation of the PCOs was 12.7 years (± ). The two groups were comparable in regard to past medical and neurological history, alcohol and drug intake. No case of a peripheral neuropathy was seen in either group. Laboratory investigations, EEG and ECG examinations revealed no clinically relevant concurrent diagnoses or group differences. Motor nerve conduction studies of the median and peroneal nerve showed no statistically significant differences. The sensory nerve action potential (NAP) of the median nerve exhibited a significantly lower mean amplitude in the group of PCOs (3.17 ± 3.11 µV versus 4.37 ± 2.64 µV, p<0.05). The corresponding slowing of median nerve sensory conduction velocity (NCV) did not reach statistical significance (p=0.1). A significant group difference could be demonstrated for the sensory NCV of the sural nerve, 47 ± 4 m/s compared to 50 ± 3 m/s in the controls (p<0.05).
In conclusion all nerve conduction studies were within normal limits. The study, however, revealed statistically significant differences concerning sensory NAP of the median nerve and sensory NCV of the sural nerve. The results are discussed in relation to their neurotoxicological and environmental health significance. Further prospective studies are needed to clarify occupational health concerns.
 

CASE-CONTROL STUDY OF POLYMORPHIC XENOBIOTIC-METABOLISING ENZYMES IN PARKINSON'S DISEASE.

G. De Palma*, A. Mutti*, P. Mozzoni*, A. Negrotti°, S. Calzetti°., *Laboratory of Industrial Toxicology, Department of Clinical Medicine, Nephrology and Health Sciences, and °Institute of Neurology, University of Parma, Parma, Italy.

"Oxidative stress" arising from exposure to xenobiotics or endogenous metabolism seems to play an important role in sporadic Parkinson’s disease (PD), but genetically-determined metabolic traits may act as effect modifiers, increasing the risk on the basis of individual detoxifying capabilities.
Methods: The distribution of polymorphic enzymes was evaluated among 100 PD (age 66,6+9,7) and 200 control patients (age 64.23+9.25), enrolled from outpatient centres of the same University Hospital. The polymorphism of the following xenobiotic-metabolising enzymes was characterised: cytochromes CYP2D6 and CYP2E1, N-acetyl transferase 2 (NAT2) and the glutathione S-transferases M1 (GSTM1), M3 (GSTM3) and T1 (GSTT1).
Results: Only the GSTT1*0 genotype only was significantly associated with PD (O.R. 1.80; 1.03-3.15). Combinations of polymorphisms occurring at multiple loci, defining metabolic haplotypes, were also evaluated. The following haplotypes, derived from defective variants of conjugating enzymes, showed a significant association with PD: GSTT1*0 / GSTM3AA (O.R. 2.69; 1.36-5.32); GSTT1*0 / NAT2 Slow Acetylator (SA) (OR 2.69; 1.34-5.40); GSTM1*0 / GSTT1*0 / GSTM3AA (O.R. 2.57; 1.11-5.96), GSTM1*0 / GSTT1*0 / NAT2 S.A. (O.R. 3.41; 1.28-9.08); GSTT1*0 / GSTM3AA / NAT2 S.A. (O.R. 4.04; 1.72-9.51), GSTM1*0 / GSTT1*0 / GSTM3AA / NAT2 S.A. (O.R. 4.82; 1.63-14.29). Interestingly, all PD patients with a deficient haplotype at the investigated conjugating enzymes were also CYP2D6*1/1 (O.R. 4.82; 1.63-14.29).
Conclusions: The combination of low penetrance genotypes - mainly belonging to conjugating enzymes - may result in a complex trait characterise by a defective scavenging capacity. Such a complex trait might increase the vulnerability of dopaminergic neurones to "oxidative stress" and subsequent neurodegeneration in PD.
 

PERIPHERAL NEUROPATHY ASSOCIATED WITH ENVIRONMENTAL EXPOSURE TO ARSENIC IN DUST.

Letz R*, Gerr F*, Ryan PB*, Green R**. *Rollins School of Public Health, Emory University, Atlanta, GA, USA; **Georgia State University, Atlanta, GA, USA.

A cross-sectional epidemiological study of peripheral neuropathy was performed among residents of a small town in Georgia, USA, who were environmentally exposed to arsenic. Unexposed comparison subjects were selected from a nearby town. To identify peripheral nerve impairment, clinical examinations and quantitative electrophysiologic and behavioral tests were performed. Because the goal was to identify cases of peripheral neuropathy, 4 clinically relevant case definitions based on examination and test results were created. Historical exposure reconstructions were performed on a subset of the exposed subjects for whom such information was available. These subjects were then rank ordered by total lifetime exposure.
Of 238 persons enrolled in the study, 133 were classified as non-exposed and 105 were classified as exposed. Subjects were excluded from analyses if they had prior occupational exposure to neurotoxicants, pre-existing conditions associated with the peripheral neuropathy, or if they were below age 18. As a result of these exclusions, 119 unexposed subjects and 84 exposed subjects were included in analyses of peripheral nerve outcomes. A total of 3 (2.5%) of 119 unexposed subjects and 12 (14.3%) of 84 exposed subjects met one or more of the case definitions for peripheral neuropathy (OR=6.4; p=0.002). An exposure-response relationship between stratified exposure level and peripheral neuropathy case status was apparent in 2 of the 4 case definitions.
These results demonstrate a strong association between environmental arsenic exposure and peripheral neuropathy among study participants. These effects occurred among people environmentally exposed to arsenic in dust rather than through more common routes of exposure such as ingestion or occupational exposure.
 

THE OCCURRENCE AND RISK ESTIMATE OF OTOTOXICITY IN CYSTIC FIBROSIS PATIENTS RECEIVING FREQUENT AMINOGLYCOSIDE THERAPY

Mulheran, M*; Degg, C*; Burr, S*; Stableforth, D**; Morgan, D**; *MRC Toxicology Unit, Leicester, UK; **Heartland’s Hospital Birmingham, UK.

Aminoglycoside ototoxicity is well recognised in clinical use, and estimates of its occurrence following single courses vary from between 5-30%. The aim of this study was to attempt to characterise in detail the occurrence and relative risk in cystic fibrosis (CF) patients; a patient group that receive repeated aminoglycoside therapy. CF patients (n=70, 10-35 yrs) had received between 1 and 53 courses of either gentamicin or tobramycin. Control subjects (n= 91) were also recruited, and both groups underwent pure tone audiometry following screening by questionnaire and tympanometry. The incidence of clinically recognised ototoxicity in all CF patients was 12/70 (17%). The median number of courses received in this group was greater than in the CF patients with no hearing loss (20 vs. 9, p=0.006). However, in patients exhibiting ototoxicity there was no clear correlation between the magnitude of hearing loss and exposure ( r_s = 0.231, p <0.4). Hearing loss in these patients was most marked over 4-8 kHz, with median thresholds falling between 20-50 dB HL. Comparison of the PTAs of both young and adult CF patients with their respective control groups showed there to be no evidence of marked sub-clinical elevations at any frequency. These results suggest that CF patients may actually be protected against aminoglycoside ototoxicity. The incidence of clinically recognised hearing loss from this study yielded an approximate risk estimate of about 1 % per course. If the condition does confer protection against aminoglycoside ototoxicity, this would have considerable implications in providing important clues to the mechanism of aminoglycoside otoxicity in man.
 

APOLIPOPROTEIN E GENOTYPE AND TOXIC ENCEPHALOPATHY

Sainio MA and Müller KMI., Department of Occupational Medicine, The National Institute of Occupational Health, Helsinki, Finland.

Organic solvents cause toxic encephalopathy in a proportion of exposed individuals. Factors which influence the individual risk of nervous system disease are unknown. The genotype of Apolipoprotein E (APOE) gene associates strongly to the risk of dementias. APOE gene’s allele e4 increases the risk of not only Alzheimer's dementia, but also vascular dementia, and memory function decline with aging. Two e4 alleles result in an additive increase and the prescence of e2 a reduction in this risk. Toxic encephalopathies due to organic solvents cause irreversible damage of the nervous system, which clinically remind subcortical dementias. The aim of this study was to reveal if APOE genotype e4 is enriched in a cohort of Finnish workers (N=98), with toxic encephalopathy after occupational exposure to organic solvents >10 years. APOE gene polymorphism at alleles e2, e3 and e4, resulting in six possible phenotypes, e2/e2, e2/e3 and e2/e4, e3/e3, e3/e4 and e4/e4, was determined by PCR from genomic DNA and HhaI-digestion.
The results demonstrate that APOE allele polymorphism in individuals with toxic encephalopathy does not differ from the normal Finnish population (Table 1). This suggests that APOE e4 does not have significant effect in the overall risk of developing toxic encephalopathy due to long-term exposure to organic solvents.
Table 1. APOE allele frequencies in toxic encephalopathy due to organic solvents.
APOE phenotype     Toxic encephalopathy (n=98)         Normal population* (n=997)
        2/2,   2/3                     7%                                                     8%
        3/3                             58%                                                     60%
        2/4,  3/4,  4/4             35%                                                     32%
*APOE phenotypes determined previously from Finnish population exposed works. Am J Ind Med 1997; 32:656-664.
 

Effects of pre- and post-natal pcb-exposure on Cognitive development in 30 month old CHILDREN

Wiener, A*; Walkowiak, J**; Fastabend, A**; Wundram, S**; Heinzow, B***; Kraemer, U**; Steingrueber, H-J*; Winneke, G**. *Institute of Medical Psychology, Heinrich-Heine University, Duesseldorf, FRG; **Medical Institute of Environmental Hygiene at the Heinrich-Heine-University, Duesseldorf, FRG; ***State Agency of Nature and Environment Schleswig-Holstein, Flintbeck near Kiel, FRG.

In a prospective study effects of environmental exposure to PCBs on mental and psychomotor development were examined. Data were collected at age 30 months (N=140) in children from the Duesseldorf area. PCB-concentrations (138, 153, 180 and S of congeners) were measured in cord blood and maternal milk (two weeks’ samples). At age 30 months, mental and psychomotor abilities were examined using the Bayley Scales of Infant Development¹ (BSID).
After control for relevant confounders no significant association was found between PCBs (S and single congeners) measured in cordblood and the BISD at 30 months, but significant negative associations occurred between PCBs in breastmilk (S of congeners) and the BSID mental and psychomotor scales at 30 months, which largely corresponds with observations in the same cohort at 7 months of age². Aditionally, negative associations between PCB congeners 138 and 180 on the one hand and the BISD scales on the other were statistically significant, too.
These results suggest, that child development at this age is negatively influenced by early developmental PCB-exposure, which covers prenatal exposure, as well. Background PCB concentrations are associated with poorer cognitive and psychomotor functioning in young children.

This work was partly supported by the EU (Brussels), Contract ENV4-CT_96-0209.

1. Bayley N. (1993) Bayley Scales of Infant Development. San Antonio, TX: Psychological Corporation.
2. Winneke G. et al. (1998) Developmental neurotoxicity of polychlorinated biphenyls (PCBs): cognitive and psychomotor functions in 7-month old children. Toxicology Letters, 102-103, 423-428.
 

Effects of pre- and postnatal pcb-exposure on Cognitive development in 42 month old CHILDREN

Wiener, A*; Walkowiak, J**; Fastabend, A**; Wundram S**; Heinzow, B***; Kraemer, U**; Steingrueber, H-J*; Winneke, G**. *Institute of Medical Psychology, Heinrich-Heine University, Duesseldorf, FRG; **Medical Institute for Environmental Hygiene at the Heinrich-Heine-University, Duesseldorf, FRG; ***State Agency of Nature and Environment Schleswig-Holstein, Flintbeck near Kiel, FRG.

Psychodevelopmental effects of environmental PCB-exposure were examined in 121 children of the Duesseldorf cohort at age 42 months. PCB-concentrations (138, 153, 180 and S of congeners) were measured in cordblood, breastmilk and 42-months plasma. Cognitive functions were assessed with the Kaufman-Assessment Battery for Children¹ (K-ABC). The K-ABC consists of the Sequential (SEQ) and Simultaneous Processing (SIM) subscales which are combined to form the Mental Processing Composite (MPC).
After control for relevant confounders, no significant associations were found between cord blood-PCBs (S and single congeners) and the K-ABC. As for breast milk, significant negative associations were found between PCBs (S , 138 and 180) and the MPC and SEQ, respectively. Borderline negative associations were found between PCBs (Sand 180) and SIM, whereas the association with PCB 138 was significant. Regarding serum from 42-months-old children, significant negative associations were found between PCBs (Sand single congeners) and the MPC. Whereas PCB138 exhibited significant negative associations with SEQ, only borderline associations were observed between PCBs (153, 180 and S of congeners) and SEQ/SIM, respectively.
These observations, besides corroborating negative associations between cognitive development and prenatal PCB-exposure as reflected in early milk-samples, are suggestive of an additional postnatal impact of background PCB-exposure, an outcome which has not been reported before.

This work was partly supported by the EU (Brussels), Contract ENV4-CT 96-0209.
Melchers P; Preuß U. (1994) K-ABC. Kaufman-Assessment Battery for Children. Deutschsprachige Fassung. Amsterdam: Swets & Zeitlinger.
 

Posters: Organophosphorus Pesticides

BIOCHEMICAL AND STRUCTURAL CHARACTERISATION OF NEUROPATHY TARGET ESTERASE

J.M. Atkins*, P. Forshaw, P.Glynn. *Medical Research Council Toxicology Unit, Hodgkin Building, University of Leicester, P.O. Box 138, Lancaster Road, Leicester, LE1 9HN (UK).

Neuropathy target esterase (NTE) is a 155kDa membrane-bound, neuronal protein, which is implicated in the initiation of organophosphorus-induced delayed neuropathy (OPIDN). NTE is also the mammalian homologue of the Swiss cheese protein which is involved in neuronal-glial cell interactions in the developing nervous system of Drosophila.
We recently cloned a 4.5kb NTE cDNA clone, D16, whose translated sequence predicts a 1327aa protein with 4 putative transmembrane domains (Lush et al., 1998). Expression of full-length D16 in mammalian COS-7 cells produces a 155kDa recombinant protein with the same substrate specificity and organophosphate sensitivity as native brain NTE. In order to establish the minimum sequence required for catalytic activity, various constructs were designed and expressed in E. coli. NEST 2F/3R (726-1215aa) produces a 55kDa, membrane-bound recombinant protein, which when reconstituted into liposomes of the synthetic phospholipid, DOPC, shows catalytic activity in hydrolysing phenyl valerate and sensitivities to OPs (mipafox, PSP, paraoxon) similar to those of native brain NTE. Shorter constructs lacking the putative transmembrane domain, TM2, do not possess catalytic activity.
TMpred analysis of NTE predicts that the active site serine (Ser-966) lies in the centre of putative transmembrane domain TM4. The formation of an oligomer containing a central pore may provide a solution for how Ser-966 could lie within a membrane and yet allow aqueous hydrolysis of the acyl enzyme intermediate. Preliminary data from patch clamp studies of active NEST 2F/3R in giant liposomes suggests NTE can form a transmembrane pore and this may give insight into its roles in normal neural development and in OPIDN.
 

SYNERGISM OF PHOSMET AND PIRIMIPHOS METHYL TO INHIBIT NEURITE OUTGROWTH FROM DIFFERENTIATING NEUROBLASTOMA CELLS

Axelrad, JC; Howard, CV*; McLean, WG. Departments of Pharmacology and * Human Anatomy and Cell Biology, University of Liverpool, Liverpool, UK.

Inhibition of outgrowth of neurites from differentiating neuroblastoma cells in vitro has been used as a model for aspects of delayed neuropathy produced by neuropathic organophosphate esters¹. It is recognised that mixtures of organophosphate compounds may have synergistic properties to inhibit protein synthesis in nerve cells to a greater extent than the individual compounds². In this study we investigated the potential of a combination of organophosphate esters to potentiate the inhibition of neurite outgrowth.
NB2a neuroblastoma cells were differentiated for 24 h in the presence of the compounds; toxicity was measured as a reduction in neurite outgrowth, which was quantified by microscopy and computerised image analysis. Phosmet and pirimiphos methyl were added individually at a concentration equivalent to the IC₂₀ (i.e. 20% inhibition of neurite outgrowth) or together in various proportions to give the same expected level of inhibition. Concentration-response curves were generated and the observed inhibition was compared with that expected if no synergism had occurred.
The combination of the two compounds produced a consistent increase in inhibition of neurite outgrowth of 64.7 (57.8 – 71.6)% (mean with 95% confidence intervals, n=7; P<0.05 Wilcoxon test). No such synergism was observed with combinations of other organophosphate compounds tested. It is concluded that a combination of phosmet and pirimiphos methyl has a greater potential for neurotoxicity than the individual compounds alone.
1. Henschler, D; Schmuck, G; van Aerssen, M; Schiffmann, D. (1992) Toxic. in Vitro 6: 327-335.
2. Marinovich, M; Ghilardi, F; Galli, CL. (1996) Toxicology 108: 201-206.
 

EFFECTS OF SINGLE AND REPEATED LOW DOSES OF ORGANOPHOSPHATES IN THE MOUSE

de Blaquière, GE; Waters, L; Blain, PG and Williams, FM. Department of Environmental & Occupational Medicine, University of Newcastle upon Tyne, UK

Organophosphates (OPs) are acutely toxic due to the inhibition of acetylcholinesterase, however, there is concern that chronic exposure may lead to long-term neurological and neurobehavioural abnormalities. We have previously shown that mipafox (a neuropathic OP), ecothiopate and paraoxon, in repeated low dosing regimens, can have a delayed effect on neurotransmission in a mouse model¹. This study investigates other currently used OPs. Single or repeated low doses of diazinon in the mouse were used to determine the relationship between acetylcholinesterase inhibition and peripheral nerve function. In vitro electrophysiological recordings were made of miniature end-plate currents (MEPPs), evoked action potentials (APs) and end-plate potentials (EPPs) in diaphragm. Maximal inhibition of acetylcholinesterase activity occurred at 3 hours after a single dose of the OP with levels remaining inhibited for 24 hours. Single low doses of diazinon inhibited blood and diaphragm acetylcholinesterase but not brain and soleus. MEPPs half-decay times were increased in a dose-dependant manner. Multiple daily dosing of OPs caused a cumulative decrease in acetylcholinesterase activity. Single doses of tested OPs caused a delayed increase in the jitter (variability of latencies) of APs but did not affect the jitter of EPPs. Repeated low doses caused an increase in EPP jitter at 28 days after dosing i.e. had a chronic effect on nerve function, although there was a threshold level for effect. Increased EPP jitter may be related to changes in conduction along the nerve and/or transmitter release. The results indicate that measurable long-term electrophysiological changes in neurotransmission can occur in the mouse following chronic dosing of OPs.

1. Kelly SS, de Blaquière GE, Williams FM & Blain PG (1997). Human & Experimental Toxicology 16, 72-78.
 

HIGH AND PERMANENT INHIBITION OF PERIPHERAL NERVE SOLUBLE ESTERASES BY MIPAFOX AND S9B, A SELECTIVE NTE INHIBITOR, SUGGEST THEIR POTENTIAL ROLE AS NEUROPATHY TARGETS

Estévez, J; García-Pérez, AG; Pellín, MC; Monroy-Noyola, A; Garis, MC; Benabent, M; Vilanova, E and Barril, J., Unidad de Toxicología. Instituto de Bioingeniería. Universidad Miguel Hernández. Alicante, España.

Phenyl valerate esterases sensitive to paraoxon, an organophosphorus compound which does not induce nor promote delayed polyneuropathy, have systematically been discarded as potential targets for neurotoxicity. Recently, however, we have shown that most peripheral nerve soluble esterases, unlike membrane-bound ones, are only transiently inhibited by paraoxon and their involvement in neurotoxic injury should therefore be reconsidered [1]. Here we show that such soluble esterases are highly sensitive to mipafox, a model neuropathy inducer. Two components were discriminated with I₅₀ values (30 min, 37 ⁰C) of approximately 13nM and 230 nM representing 53% and 40% of total activity, respectively. The second order rate constant (k_a) for the most sensitive component was about 1.2 x 10⁶ M^-1. min^-1. A selective neuropathy target esterase (NTE) inhibitor, S9B, also inhibited more than 80% of these esterases with a k_a of about 3.1 x 10⁶ M^-1. min^-1. Both mipafox and S9B inhibitions seem to be permanent ones as they are not reverted after inhibitor removal by ultrafiltration. The above findings suggest a possible role for peripheral nerve soluble esterases as neuropathy targets.

[1] J. Barril*, J. Estévez, M.A. Escudero, M.V. Céspedes, N. Ñíguez, M.A. Sogorb, A. Monroy and E. Vilanova, Peripheral nerve soluble esterases are spontaneously reactivated after inhibition by paraoxon: implications for a new definition of Neuropathy Target Esterase, Chem.-Biol. Interact. (1999) in press.
 

IS THE DEVELOPMENT OF ORGANOPHOSPHATE-INDUCED DELAYED NEUROPATHY (OPIDN) DUE TO A GAIN OF FUNCTION IN NTE?

Yong Li, David J Read and Paul Glynn. MRC Toxicology Unit, Leicester, UK.

In adult chickens, a dose of neuropathic OP causing >70% inhibition of brain NTE leads, after a delay of 1-2 weeks, to axonal degeneration causing limb weakness and paralysis. NTE inhibitors, including some organophosphinates, which do not generate a negative charge at the active site serine are not neuropathic, even following sustained inhibition of NTE by repeated dosing. The esterase function of NTE therefore appears redundant in these birds. Thus, either: 1. Neuropathic OPs cause loss of a non-esterase function of NTE which is required by neurons and/or their axons, or; 2. NTE has no essential role in the adult chicken, but neuropathic OPs induce a toxic gain of function.
There is a general correlation, across a range of vertebrate species, between low levels of brain NTE activity and resistance to OPIDN. Humans and chickens have a brain NTE activity of > 2000 nmoles/g brain/min while rats and mice have only 40% and 30% respectively, of this level of activity. Furthermore, NTE turns over more rapidly (t_1/2= 2d) in mice than in chickens (t_1/2= 4-5d). Neither rats nor mice developed OPIDN following a single administration of neuropathic OP despite an initial NTE inhibition of >90%. We have dosed adult male C57Bl/6J mice daily with mono-ortho-cresyl diphenyl phosphate so that brain NTE inhibition was maintained above 85% for 21 days with no clinical signs of OPIDN. By contrast, mice are not generally resistant to toxic neuropathies since dosing with a non-OP, bromophenylacetylurea, produced unambiguous hind-limb weakness.
The above suggest that initiation of OPIDN may require an absolute level of neuropathic OP-modified NTE to be maintained for a certain period. We are now attempting to generate transgenic mice with over-expression of NTE in neural tissue; will these animals be susceptible to OPIDN?
 

DIFFERENTIAL EFFECTS OF CHLORPYRIFOS OXON AND PARAOXON ON MUSCARINIC AUTORECEPTOR FUNCTION IN VITRO.

Liu, J, and Pope, C. Division of Toxicology, Northeast Louisiana University, Monroe, Louisiana, USA.

Chlorpyrifos oxon (CPO) and paraoxon (PO) are the active metabolites of the organophosphorus pesticides chlorpyrifos (CPF) and parathion (PS). Some studies suggest that similar levels of acetylcholinesterase (AChE) inhibition by these pesticides can result in markedly different degrees of cholinergic toxicity. Acetylcholine (ACh) release and its modulation by muscarinic autoreceptors (muscarinic autoreceptor function, MAF) are differentially affected by CPF and PS in vivo. ACh release can be studied by preloading brain slices with [³H]choline and stimulating with high-potassium buffer containing hemicholinium-3 (to prevent re-uptake of radiolabeled choline). Under these conditions, muscarinic receptor agonists (e.g., carbachol, cis-dioxolane) and AChE inhibitors (e.g., eserine) reduce ACh release in an atropine-sensitive manner by direct activation of muscarinic autoreceptors or indirectly by elevating endogenous ACh, respectively. Thus, any direct action of an anticholinesterase on muscarinic autoreceptors can be masked by its indirect activation of MAF via endogenous ACh. We studied effects of CPO and PO on ACh release under two different conditions: 1) in the absence of either eserine or atropine in the perfusion buffer or 2) in the presence of both. When added in the absence of eserine and atropine, both agents reduced release in a concentration-dependent manner (24-31% at 0.1-10 m M), similar to agonists, carbachol and cis-dioxolane. In the presence of eserine (20 m M) and atropine (0.1 m M), the relative potencies of all four agents were reduced. PO decreased release as before (42% reduction at 1 mM) but CPO increased ACh release (36% at 0.3 mM). We conclude that CPO and PO have both direct and indirect actions on MAF. Differences in direct actions at the muscarinic autoreceptor could contribute to differential toxicity with these toxicants.
 

ANTICHOLINESTERASE ACTION OF FLUORINE CONTAINING AMINOPHOSPHONATES.

Makhaeva, GF, Malygin, VV, Zhuravleva, LV, Aksinenko, AYu, Sokolov, VB, Razdolsky, AN. Institute of Physiologically Active Substances Russian Acad. Sci., Chernogolovka 142432, Russia

A series of O,O-dialkyl-1-phenylsulfonamido-1-trifluoromethyl-2,2,2-trifluoroethyl phosphonates of general formula (RO)₂P(O)C(CF₃)₂NHSO₂C₆H₅, where R = Et, Pr, iPr, Bu, iBu, Pent, iPent, Hex, has been tested on the inhibitory effect on human erythrocyte acetylcholinesterase (AChE, EC 3.1.1.7)) and horse serum butyrylcholinesterase (BuChE, EC 3.1.1.8.). The compounds studied except R=iPr were found to be strong irreversible inhibitors of both esterases: k_i(AChE)=4.14x10⁴ to 1.29x10⁶M^-1min^-1, k_i(BuChE)=1.0x10⁶ to 9.76x10⁶M^-1min^-1, antiesterase activity increased with hydrophobicity of R. Whereas a -branching in alkoxy substituent (R=iPr) drastically reduces antiesterase activity: k_i(AChE)=1x10¹M^-1min^-1, k_i(BuChE) = 2.15x10² M^-1min^-1. X-ray analysis of the phosphonate with R=iPent has shown the considerable lengthening of covalent bond P-C(sp³) up to 1.888 Å in comparison to standard length 1.844 Å . This fact as well the presence of two electron withdrawing CF₃ groups could be the reason for shortening the P-C(sp³) bond and its easy cleavage. These data along with data on structure-antiesterase activity relationships allow to suggest that cholinesterase inhibition by the studied aminophosphonates is going with cleavage of P-C(sp³) bond.

The research was supported by RFBR, Grant # 98-04-48831.
 

O,O-DIALKYL-O-(1-CARBOETOXY-2,2,2-TRIFLUOROETHYL) PHOSPHATES AS INHIBITORS OF NEUROPATHY TARGET ESTERASE AND ACETHYLCHOLINESTERASE.

Malygin V., Makhaeva G., Zhuravleva L., Goreva T., Aksinenko A., Sokolov V., Institute of Physiologically Active Substances Russian Acad. Sci., Chernogolovka 142432, Russia

The inhibitor potency of a series of phosphates (RO)₂P(O)OCH(CF₃)COOC₂H₅ (DCFP, where R= Me, Et, Pr, iPr, Bu, iBu, Pent) against hen brain neuropathy target esterase (NTE) and acetylcholinesterase (AChE) has been examined to provide new data on inhibition specificity of these neurotoxicologically significant esterases and to assess the potential of DCFP to cause organophosphate induced delayed neurotoxicity (OPIDN). Compounds studied were found to inhibit irreversibly both esterases, log k_i values were in the region 1.20 to 4.93 for NTE and 2.62 to 4.32 for AChE. DCFP with short R (Me, Et) were shown to be far more potent inhibitors of AChE than NTE. Both anti-NTE activity, selectivity for NTE and, correspondingly, the propensity of compounds to cause OPIDN rise with increasing their hydrophobicity. A high value of k_i(NTE)/k_i(AChE) = 4.07 for R = pentyl suggests that this compound would have the potential to cause OPIDN at doses lower than the LD₅₀.
With multiple regression analysis (Hansh’s models) structure-antienzymatic activity relationships were investigated, k_i and Michaelis constants, K_a, were analyzed. The contribution of hydrophobic interactions to NTE inhibition was shown to be more significant than that to AChE inhibition. The results of QSAR modelling for K_a(NTE) demonstrated that the influence of hydrophobic interactions and steric hindrances are revealed at the sorption stage and so determine the inhibitor reactivity of the compounds. A slope equal to one descriptive of the dependence of lgK_a(NTE) on hydrophobicity confirms the extractive mechanism for NTE interaction with organophosphorus inhibitors.

The research was supported by RFBR, Grant # 98-04-48831.
 

HYDROLYSIS OF HEXYL DICHLOROPHENYL PHOSPHORAMIDATE IN HEN, RAT AND RABBIT.

Monroy-Noyola A; Sogorb MA; Ñíguez N; Estévez J; García-Pérez AG; Barril J; Garis M; Benabent M; Pellín MC and Vilanova E., Unidad de Toxicología, Instituto de Bioingenieria. Universidad Miguel Hernández. Alicante, España.

The O-hexyl O-2,5-dichlorophenyl phosphoramidate (HDCP) is a chiral compound analogous of methamidophos insecticide. This racemic mixture induce delayed neuropathy in hen. In this study we have characterised the hydrolysis of HDCP regarding its Ca²⁺-dependence, EDTA-resistance and stereospecific activities in serum and subcellular fractions (particulate and soluble) arising from kidney, brain and liver of hen, rat and rabbit using a colorimetric microassay. The results show a significant stereospecific Ca²⁺-dependent hydrolysis of HDCP (S-HDCP > R-HDCP) in serum and particulate fractions of liver and kidney from rat and rabbit. The particulate fractions of brain in the three studied species and hen serum were slightly stereospecifics. On contrary, the EDTA showed low levels of HDCP hydrolysis with exception of hen serum. In some fractions the EDTA-resistant activity was slightly stereospecific (R-HDCP > S-HDCP). The present in vitro study reinforces the idea that R-HDCP is the isomer that ultimately survives in vivo after the hydrolysis of HDCP and could be the isomer responsible of the neurotoxicity of this racemic compound.

Acknowledgements: Work supported by grant SAF96/0168 (CICYT, España). A Monroy received a fellowship from ICI-AECI and CONACYT.
 

THE ROLE OF METABOLISM IN DETERMINING SUSCEPTIBILITY TO ORGANOPHOSPHATE TOXICITY IN MAN

Mutch, E., Blain, PG. and Williams, FM. The Toxicology Unit, Environmental and Occupational Medicine, Newcastle University, NE2 4HH, UK.

Individuals vary widely in their toxic response to phosphorothioate pesticides. This may be influenced by the individuals’ capacity to activate and detoxify these compounds. In this study the capacity to metabolise chlorpyrifos (O,O-diethyl O-3,5,6-trichloro-2-pyridyl phosphorothioate) was studied in human liver.
Human liver microsomes (0.25mg protein) were incubated at 37oC with chlorpyrifos (100µM) or its active metabolite, chlorpyrifos oxon (500µM), for 1min or 2.5min, respectively. Formation of 3,5,6-trichloro-2-pyridinol (TCP) and chlorpyrifos oxon (CPO) were determined. Liver microsomes were also pre-incubated (10min) with classical CYP3A inhibitors (100µM). Microsomes (20pmol P450) from cell lines expressing individual human CYPs were incubated with chlorpyrifos (100µM) and formation of CPO and TCP determined. CYP3A (nifedipine oxidation) and A-esterase (paraoxon hydrolysis) activities were also measured.
Chlorpyrifos was metabolised to TCP with a 4-fold range in activity (1.32-4.86 nmol/min/mg protein, n=7) but no oxon was detected. TCP was produced from chlorpyrifos oxon (70.8-221.7 nmol/min/mg protein). CYPs 3A4, 3A5 and 2C8 produced TCP from chlorpyrifos (13.1, 28.5 and 18.0pmol/hr/pmol P450, respectively) but no oxon was detected. The CYP3A inhibitors reduced formation of TCP from chlorpyrifos by approx. 50%. TCP formation from chlorpyrifos and TCP formation from CPO were significantly correlated (p<0.05). TCP formation from chlorpyrifos and p-nitrophenol from paraoxon were also significantly correlated (p<0.05) but TCP formation from chlorpyrifos and nifedipine oxidation were not.
These results indicated the involvement of CYPs 3A and 2C8 in chlorpyrifos metabolism, similar to parathion. However, A-esterase hydrolysis was more important for chlorpyrifos metabolism than previously shown for parathion¹. Variability in A-esterase activity relates to expression of the R and Q variants of PON1² and this is currently being investigated in human liver.

1. Mutch E, Blain PG and Williams FM (1999). Toxicol Lett (in press).
Davies HG, Richter RJ, Keifer M, Broomfield CA, Sowalla J and Furlong CE (1996). Nature Genet 14: 334-336.

DETECTION AND ANALYSIS OF PREVIOUSLY UNRECOGNISED TARGETS FOR ORGANOPHOSPHORUS COMPOUNDS.

Richards, P. & Lees, J.* MRC Toxicology Unit, Hodgkin Building, Lancaster Road, Leicester, LE1 9HN, UK. *Space Research Centre, Department of Physics and Astronomy, University of Leicester, LE1 7RH, UK.

There have been a number of reports that low level exposure to organophosphorus (OP) compounds may lead to adverse health effects in humans, especially neuropsychological problems. We have undertaken a mechanistic investigation of the above issue by searching for potential targets of organophosphorus compounds in the CNS. Our methods involve screening for targets by employing radiolabelled OP ³H-diisopropylflurophosphate) to produce a profile of serine-hydrolases in rat brain. Electrophoresis (both 1 and 2D) is used to survey proteins in terms of pI and molecular mass enabeling a proteomics approach to target identification. Pre-incubation with ‘cold’ OP pesticides can be used to block DFP labelling allowing the relative sensitivity of targets to be determined and thus their toxicological relevance assessed. Because of the low abundance of DFP-sensitive proteins in the brain (< 0.07% of total brain protein will react with 10mM DFP) we have employed a novel microchannel-plate detector system which boasts a spatial resolution of 70mM, a linear dynamic range of at least 6 orders of magnitude and a minimum ³H detectable activity of < 60 dpm /mm². A surprising target in the brain that has been discovered during our survey is the enzyme acylpeptide hydrolase (ACPH). This peptidase, which catalyses the hydrolysis of N-acylated amino acids from short peptides, is highly sensitive to certain OP compounds. The exact biological function of ACPH is unknown but an interesting structure-activity relationship between this enzyme and a putative non-cholinergic, cognitive-enhancing effect of certain OPs is observed.
 

CHOLINERGIC AND MONOAMINERGIC EFFECTS OF CARBARYL IN RATS

Sachana, M*; Flaskos, J*; Nikolaidis, E**; Hargreaves, AJ ***; Alexaki, E*. *Laboratory of Biochemistry and Toxicology, and **Laboratory of Pharmacology; Faculty of Veterinary Medicine, Aristotelian University of Thessaloniki, Thessaloniki, Greece; *** Department of Life Sciences, The Nottingham Trent University, Nottingham, UK.

Carbaryl (CB), the most widely used carbamate pesticide, was studied in rats in terms of its effects on (i) the activity of brain acetylcholinesterase (AChE), (ii) the activity of plasma cholinesterase (ChE), (iii) the activity of brain monoamine oxidase (MAO), and (iv) the rate of uptake of 5-hydroxytryptamine (5-HT) by the platelets. CB, dissolved in a 3:1 propylene glycol:water solution, was administered to adult, male rats intraperitoneally either in a single dose of 50% LD₅₀ (50 mg/kg) or in a repeated dose of 10% LD₅₀ (10 mg/kg) daily for 15 days. The acute dose of the drug produced a significant (p<0.001), 54 percent mean inhibition of brain AChE activity 0.5 h after CB administration. Plasma ChE activity was also significantly (p<0.05) inhibited under these conditions. In CB-treated rats a trend towards a decrease in the activity of brain MAO activity was noted. In these animals the rate of active (clomipramine-inhibitable) uptake of 5-HT by the platelets was not significantly different from controls. In the repeated dosing experiment, CB was found to cause slight, not statistically significant reductions in both brain AChE and plasma ChE activities 24 h after administering the last dose. This dosage regimen had no significant effects on brain MAO activity and platelet 5-HT uptake.
 

NEUROPATHY TARGET ESTERASE ACTIVITY IN BLOOD: BIOSENSOR ANALYSIS

Sigolaeva, LV¹; Makower,A²; Eremenko, AV³; Makhaeva, GF⁴; Malygin, VV⁴; Kurochkin, IV^1,3; Scheller F²., 1 Faculty of Chemistry, M.V. Lomonosov Moscow State University, Moscow, 119899 Russia; 2 Department of Analytical Biochemistry, Potsdam University, Im Biotechnologiepark, 14943 Luckenwalde, Germany; 3 Research Center for Molecular Diagnostics and Therapy, Moscow, 113149 Russia; 4 Institute of Physiologically Active Substances, Russian Academy of Scienses, Chernogolovka, 142432 Russia

Neuropathy target esterase (NTE) is a specific target for neuropathic organophosphates (OP) and it seems to be a site of initiation of OP-induced delayed neuropathy (OPIDN). NTE inhibition within hours of exposure to OP predicts potential for the development of OPIDN. Fast and effective methods monitoring of neuropathic OP exposure to humans are highly required.
New biosensor method for the analysis of NTE and its inhibitors is based on the combination of NTE enzymatic hydrolysis of phenyl valerate with phenol detection by carbon-paste tyrosinase electrode. The use of the tyrosinase electrode leads to 10-fold improvement in the sensitivity of the analysis of NTE activity in comparison with the spectrophotometric detection. The tyrosinase electrode was found to be suitable for measurements of NTE activity in whole human blood when the application of the usual spectrophotometric detection is considerably limited. The results of inhibitor specificity obtained for mipafox and DFP titration of NTE in blood were close to those for neuronal NTE. NTE-like activity in blood was found to be equal 0.20 nmoles/min per mg of protein.

The research was supported by Russian Foundation for Basic Research, Grant # 98-04-48831 and the German Bundesministerium fur Bildung, Wissenschaft, Forschung und Technologie, Grant FKZ 0311684
 

THE EFFECT OF SOMAN ON PRODUCTION OF THE CYTOKINE INTERLEUCIN-1 b IN RAT BRAIN.

Svensson, I; Waara, L; A Bucht; Cassel, G. Defence Research Establishment, Division of NBC Defence, Department of Biomedicine, SE-901 82 Umeå Sweden

Interleukin-1b is one of several cytokines involved in inflammation both outside and inside CNS. It also plays a role in noninflammatory processes in CNS, e.g. downmodulation in pain and stimulation of hypothalamic hormone release. Another function is stimulation of astroglial proliferation and it is shown that IL-1b regulates amyloid precursor protein mRNA expression. In CNS IL-1b is produced by microglia and astrocytes.
We have, in previous studies, shown pronounced effects after soman intoxication in dopaminergic, GABAergic and cholinergic systems in rat brain. Here we report on inflammatory response indicated as IL-1b , after soman intoxication.
The effect of soman on production of IL-1b was measured by ELISA. Two hours after administration of The LD₅₀ dose soman (100 µg/kg) we observed a moderate increase in IL-1b production in the frontal cortex, which was followed by a marked increase six hours after soman intoxication. In different experiments there was a variation in the amplitude of the effect at six hours. This could be due to the steep toxic curve of soman, which has been shown in previous experiments (unpublished results), in combination with the differences in physiological responses in rats. The high levels of IL-1b appeared to be related to the occurrence of seizures, since rats treated with 100 µg/kg soman and did not have seizures, had no increased levels of the cytokine.
The study also involves gene expression (RT-PCR) to further elucidate the effects of soman on the IL-1b response in CNS.

Giulian D; Lachman LB. (1985) Science 228: 497-499.
Grierson JP; Petroski RE; Ling DSF; Geller HM. (1990) Dev Brain Res. 55:11.
Benveniste, EN. (1993) Astrocytes: Pharmacology and Function, S Murphy, ed., Academic Press, San Diego, CA, pp.355-382..
 

Posters: Miscellaneous Toxicants

DIFFERENTIAL EFFECTS OF ORALLY VERSUS PARENTERALLY ADMINISTERED QINGHAOSU DERIVATIVE ARTEMETHER IN DOGS

W. Classen, B. Altmann, P. Gretener, C. Souppart, P. Skelton-Stroud, G. Krinke, Novartis AG, CH-4332, Stein, Switzerland.

Artemether (AM) is an antimalarial drug derived from artemisinin (Qinghaosu), an extract of the herb Artemisia annua L., sweet wormwood. Its antiparasitic effect is that of a schizontocide and is explained by rapid uptake by parasitized erythrocytes and interaction with a component of hemoglobin degradation resulting in formation of free radicals. It has been shown to exhibit a high clinical cure rate. Previous animal safety studies with Qinghaosu derivatives revealed dose-dependent neurotoxicity with movement disturbances and neuropathic changes in the hindbrain of intramuscularly treated dogs, rats and monkeys. Such effects have not been seen in man.
In a pilot study 20 mg/kg/day of AM was given i.m. to groups of 3 male Beagle dogs for 5 and 30 days, respectively. Clinical signs of neurotoxicity were noted in some individual dogs from test day 23 on. One dog had to be sacrificed pre-term. Microscopic examination demonstrated neuropathic changes only at 30 days, but not at 5 days. Animals had neuronal and secondary axonal damage, most prominent in the cerebellar roof, pontine and vestibular nuclei, and in the raphe/paralemniscal region. The affected neurons showed loss of Nissl substance, cytoplasmic eosinophilia, shrinkage of the nucleus and in advanced stages scavenging by microglia.
In a subsequent experiment, AM was administered to groups of 4 male and 4 female dogs, respectively, at 8 daily doses of 0, 20, 40 or 80 mg/kg i.m., or 0, 50, 150 or 600 mg/kg p.o. Neurologic signs were seen at high i.m. doses only. In most animals they were inconspicuous and consisted of reduced activity with convulsions seen in single dogs shortly before death. Neuronal damage occurred in all animals at 40 and 80 mg/kg following i.m. treatment. At 20 mg/kg minimal effects occurred in 5/8 dogs only, indicating that this level was close to tolerated exposure. No comparable lesions were observed after oral administration.
I.m. administered AM indicated peak plasma levels of AM at 2 to 4 hours post-dose, slow elimination and a tendency to accumulate after repeated administration. This plasma profile is comparable to that observed in humans after oral or parenteral administration. AM levels in the cerebrospinal fluid (CSF) were < 10% of plasma levels. After oral administration AM concentrations were considerably lower than after i.m. administration on day 1 and almost nil on day 7 indicating its fast inactivation in dogs.
 

EXPRESSION OF HEAT SHOCK PROTEIN 72 IN BRAIN STEM LESIONS

Davidson, G., Guérin, C., Nolan, C.C. & Ray, D.E. MRC Toxicology Unit, Hodgkin Building, Lancaser Rd. Leicester, LE1 9HN.

Heat Shock Protein 72 (HSP 72) is induced in cells that are experiencing stress. 1,3-Dinitrobenzine (DNB) and S-alpha Chlorohydrin (Chlorohydrin) are selective neurotoxins which interfere with different components of the cellular energy pathway. This study was designed to identify which cell type(s) express HSP 72 after exposure to DNB or Chlorohydrin.
Young adult rats were given neuropathic doses of DNB (3 x 10mg/kg) or Chlorohydrin (160mg.kg). Animals were killed by transcardiac perfusion at 1, 3 and 8 days, the brains were removed embedded in paraffin, and sectioned at 10 µm. Sections were incubated with antibodies to HSP 72 alone or in combination with antibodies to glial fibrillary acidic protein (GFAP), ED1, or neurofilaments (NF) to identify cell types.
HSP 72 co-localized with GFAP and ED1 indicating that some of the staining observed was in glial cells and macrophages. Vascular labelling was seen both within the lesioned areas and in adjacent unaffected brain areas and appeared to be localized within the vascular endothelial cells. While some vessels expressed HSP 72 quite strongly, nearby vessels had no detectable immunolabelling.
Double-label immunocytochemistry clearly demonstrated positive HSP 72 immunolabelling in surviving astrocytes within 8 day brain stem lesions. Macrophages were also HSP72 positive, which could be either endogenous or as a result of phagocytosis of cellular debris. Positive immunolabelling of vascular endothelium, even well away from the active lesion site, would indicate that both DNB and Chlorohydrin have a direct affect on this cell type. The expression of HSP 72 by surviving glial and vascular cells within lesioned areas could indicate that expression of this protein may confer a protective effect.
 

POLYCHLORINATED BIPHENYLS INCREASE INTRACELLULAR CALCIUM THROUGH PURINERGIC RECEPTOR ACTIVATION IN HUMAN MACROPHAGES

Dehnhardt, M; Desaiah, D; Wiegand, H; Medical Institute of Environmental Hygiene, Heinrich-Heine-University, Dusseldorf, Germany.

Polychlorinated biphenyl compounds (PCBs) are environmental neurotoxicants shown to exert their toxicity through increased intracellular calcium [Ca²⁺]_i in n. number of cells. The mechanism of the disruption of calcium homeostatis by PCBs is not well understood. However, the non-planar but not co-planar PCI3s were shown to disrupt calcium dependent signalling pathways. Purinergic receptors such as P2X and P2Y are known to regulate the [Ca²⁺]_i by either ligand gated ion channel or through (G-protein coupled receptor activation mechanisms, Aclenosine trisphosphate (ATP) is a natural ligand for many of the sub-types of purinergic receptors including P2X and P2Y. Human macrophages are known to express both P2XC₇ and P2Y₂ receptors. Therefore, our objective of this study was to determine whether PCBs enhance the [Ca²⁺]_i in human macrophages and, if so, is it due to the activation of purinergic receptors.
Human monocyte derived macrophage cultures were treated with different concentrations of ATP, PCBs, specific receptor blockers such as suramin and XAMR. The [Ca²⁺]_i imaging was conducted using Fura-2 as fluorescent probe. The results obtained showed that the [Ca²⁺]i was increased as a function of ATP concentration with an EC₅₀ of 7.1 m M. The ATP mediated increase of [Ca²⁺]_i was blocked by both suramin and XAMR which are known antagonists of P2Y₂ receptors. 2,2’,4,4’-tetrachlorobiphcnyl, a non-planar PCB increased the ATP mediated [Ca²⁺]i as evidenced by a shift of ATP dose-response curve to the left reducing the EC₅₀ of ATP from 7.1 to 1.5m M. XAMR, the P2Y₂ receptor antagonist, blocked the PCB effect. 3,3’, 4,4’-tetrachlorobiphcnyl, a co-planar PCB, had no effect on purinergic receptor activity. These data clearly demonstrate for the first time that non-planar PCBs increase [Ca²⁺]_i by the modulation of purinergic receptors in human macrophages.
 

CHRONIC EXPOSURE TO LITHIUM ENHANCES CHOLINERGIC ACTIVITY IN THE FRONTAL CORTEX AND THE HIPPOCAMPUS

Finkelstein, Y. Division of Diagnostic Neurology, Department of Neurology, Shaare Zedek Medical Center, Jerusalem, Israel.

The rate of choline uptake by the presynaptic neuron regulates the cholinergic activity during prolonged periods of continuous emotional, hormonal and nervous stimulation¹. This is expressed by the selective decrease in choline uptake and the increase in acetylcholine release in presynaptic cholinergic terminals, following prolonged stress periods of hours or days. In a like manner, long-term low-level exposure to divalent lead caused enhanced cholinergic activity in the septohippocampal systems². Lithium, a monovalent cation, is known for its neuromodulatory properties. Lithium chloride is used as a therapeutic agent for mood stabilization and is administered over extended periods. The present study was aimed to determine the cholinergic effects of chronic exposure to lithium.
Two-month-old female Sprague-Dawley rats were fed for six weeks with a diet containing lithium chloride. Therapeutic synaptosomes were prepared from dissected rat brain tissues. Using radioisotopic tracers, the parameters of cholinergic activity were measured in the synaptosomal preparations from rat hippocampus and frontal cortex. Presynaptic choline uptake (following 10 min. incubation at 37° C), presynaptic acetylcholine release and postsynaptic quinuclidinyl benzylate binding were measured.
Lithium treatment resulted in plasma levels of 0.8 mEq/L similar to the levels observed in treated patients. Increased levels of choline uptake and acetylcholine release were observed in both hippocampal and frontal cortex synaptosomes. Choline uptake increased by 24% over control levels after 10 min. incubation. The release of newly synthesized acetylcholine from hippocampal synaptosomes was increased by 35%. This increase was not observed in frontal cortex synaptosomes. Maximal quinuclidinyl benzilate binding (B_max) by both hippocampal and frontal cortex homogenates was increased by 42% and 43% respectively, with no change in K_d values.
In conclusion, chronic exposure to lithium enhanced all parameters of cholinergic activity in rat brain. However, the effect of lithium differed from the pattern of lead-induced cholinergic activation. To some extent the lithium effects mimicked the stress-induced cholinergic changes. This finding concurs with the therapeutic role of lithium as a mood stabilizer.
1. Finkelstein et al. (1985) Brain Research 343:314-319.
2. Finkelstein et al. (1998) Brain Research Reviews 27:168-176.
 

GLIAL-VASCULAR INTERACTIONS FOLLOWING METABOLIC NEUROTOXINS

Guérin, C., Davidson, G., Nolan, C.C. & Ray, D.E. MRC Toxicology Unit, Hodgkin Building, Lancaser Rd. Leicester, LE1 9HN.

It is generally accepted that glial cells are necessary for the formation of the blood brain barrier (bbb) during development. However, there is little evidence relating to their function in maintenance or repair of the barrier in the adult. The metabolic neurotoxins 1,3-Dinitrobenzine (DNB) and S-alpha Chlorohydrin (Cholorhydrin) both produce glial lesions and bbb breakdown although their precise mechanism of action differs. This study used multiple antibody immunocytochemistry and intraveinous tracers to more clearly define the interactions between glial and vascular cells in these neurotoxic lesions.
Rats were given neuropathic doses of DNB (3 x 10mg/kg) or Chlorohydrin (160mg.kg) and killed from 6 hours to 8 days later by perfusion fixation. Blood brain barrier breakdown was visualized by giving intraveinous tracers 1 hour before perfusion. Brains were sectioned at 80µm and processed for confocal immunocytochemistry. Antibodies to glial fibrillary acidic protein (GFAP), or neurofilaments (NF) were used as markers of glia and neurons, respectively.
Barrier breakdown was an early event with both agents and coincided with glial degeneration. Surprisingly, astrocytes contacting blood vessels appeared less susceptible to the toxins than those in the surounding neuropil. At intermediate time-points severely affected animals had large hemmorhagic lesions although many of the peri-vascular astrocytes still appeared intact. At 8 days large glial scars had formed but vessels within the lesioned areas showed no evidence of active leakage. While some vessels within the lesion appeared to have retained their glial ensheathment others were clearly denuded of all glial contact.
These results would support the hypothesis that glial-vascular contact is unnecessary for the maintenance of the adult bbb. It remains to be determined if glial cells are involved in the repair of the leaking bbb in these neurotoxic lesion models.
 

THE ROLE OF VASCULAR DAMGE IN THE PATHOGENESIS OF CHEMICAL ENCEPHALOPATHIES

Lister, T., Nolan, C.C., Guérin, C.J. & Ray, D.E., MRC Toxicology Unit, Lancaster Road, Leicester LE1 9HN, U.K.

Vascular damage can play an early and a prominent role in the pathogenesis of brain lesions, but is often overlooked in studies of neurotoxicity. This is in part due to its transient nature. Vascular damage is an early feature of the lesions produced by nitrobenzene, dinitrobenzene¹, metronidazole, and nitropropionic acid², and appears to be a later feature of the damage produced by a -chlorohydrin. The protein leakage and petechial haemorrhage associated with vascular breakdown is less easily missed if thick sections, immunostaining for plasma protein, or exogenous vascular tracers are used. Functional measures are also of value, dinitrobenzene producing a large, early, and regionally specific increase in blood flow, which accompanies the transiently increased vascular permeability¹. This flow increase is, at least in part, mediated by nitric oxide. The nitric oxide synthesis inhibitor L-NAME (20 mg/kg i.p.) given at 4 hours after the last of 4 daily intraperitoneal doses of 7.5 mg/kg dinitrobenzene, significantly reduced the blood flow increase seen at 24 hours from 222.6 ± 53.5% (n=6) to 106.0 ± 17.1% (n=8) in the inferior colliculi of male F344 rats (mean ± S.E. as % of predose blood flow). Blood flow was measured by hydrogen polarography. This result shows that the vascular component of the lesions can be manipulated pharmacologically, and opens a path to investigation of neuro-vascular pathogenesis.
¹ Romero, I., Brown, A.W., Cavanagh, J.B., Nolan, C.C., Ray, D.E. & Seville, M.P. (1991) Neuropath. Appl. Neurobiol. 17: 495-508.
² Nishino, H., Shimano, Y., Kumazaki, M., and Sakurai, T. (1995) Neurosci. Lett. 186:161-164.
 

THE EFFECTS OF QUININE ON COCHLEAR NERVE FIBRE ACTIVITY IN THE GUINEA PIG

M. Mulheran, MRC Toxicology Unit, Leicester, U. K. LE1 9HN.

The ototoxicity of quinine is well documented and is associated with plasma levels of quinine between 7-20 ug ml ^-1. This study aimed to establish what physiological changes at the level of the auditory nerve correlated with these plasma levels. Single unit electrophysiological recordings (n=38) in four neuroleptanaesthetised guinea pig were carried out as described elsewhere¹. During recording the animals body temperature, end tidal CO₂ and systemic blood pressure were kept within normal limits. Between 10-30mg/kg of quinine i.v. was injected slowly. Data were analysed using Students t-test and the chi-squared test.
The frequency tuning curves (FTCs) of these fibres exhibited significant increases in the thresholds of both ‘tip’ and ‘tails’ regions of the FTC, but these changes did not appear to be accompanied by significant changes in sharpness of tuning, as measured by the Q_‘10’dB. In comparison with control fibres (n=178) from 13 untreated animals, significant changes in the proportion of low:high spontaneous rates (SR) were also seen. Using a boundary criterion of 25 sp/s, this changed from 26%:74% to 47%:53% in control and quinine poisoned fibres respectively and was significant at p<0.01. Independent of changes in SR, significant increases (p<0.005) in the mean absolute refractory period (ARP) from 0.85 ms to 1 ms were measured following quinine administration. The absence of a significant effect on the fibre tuning whilst threshold was elevated indicates that; quinine acts at more than one site in the cochlea, does not affect the integrity of the cochlear amplifier, but does appear to affect cochlear sensitivity.

1. Mulheran, M. (1999). Hearing Research in press.
 

CAN CHOLINERGIC LESIONS PREVENT MK-801-INDUCED NEUROTOXICITY IN THE RAT CINGULATE CORTEX?

Willis, CL^*, Ray, DE^*, Kind, CN^**, Marshall, H^**, Pearson, EC^**, Evans, JG^** and Hammond, TG^**. ^*MRC Toxicology Unit, University of Leicester, LE1 9HN; ^**Safety Assessment, Astra Charnwood, Loughborough, LE11 5RH, U.K.

Glutamate receptor antagonists have been shown to reduce excitotoxic neuronal injury. However, in layers III/IV of the posterior cingulate and retrosplenial cortex several NMDA receptor antagonists are neurotoxic: inducing cytoplasmic vacuoles and expression of stress protein Hsp72. To explain this paradox, Olney has proposed that these antagonists inhibit GABAergic neurones, reducing inhibition of a tonic cholinergic stimulation of cingulate neurones, thus resulting in cholinergic hyperstimulation and pathological changes. Therefore, a lesion of the cholinergic input to the cingulate may affect NMDA antagonist-induced vacuolation. Female Fisher rats, 12 weeks of age, received a unilateral injection in the basal nucleus of Meynert of either 192-IgG-saporin (80 ng), a highly selective cholinergic immunotoxin, or phosphate buffered saline. Seven days later they were given a subcutaneous injection of MK-801 (5 mg/kg) or saline. After a further 6-27 h, the brains were fixed in buffered 4% paraformaldehyde and processed for assessment of vacuole formation and immunohistochemical detection of choline acetyltransferase (ChAT) immuno-reactivity and Hsp72 mRNA. 192-IgG-saporin treatment resulted in a large reduction in ChAT immunoreactive cells in the ipsilateral nucleus of Meynert compared to the uninjected contralateral nucleus. Six hours after MK-801 administration the contralateral posterior cingulate pyramidal layer showed a marked increase in Hsp72 mRNA expression which was largely absent in the ipsilateral cingulate and in 192-IgG-saporin treated rats dosed with saline. These results go some way to support the hypothesis that cholinergic input to the cingulate is an important factor in influencing NMDA antagonist neurotoxicity.