Mathematical modelling and quantitative methods.

The present review reports on the mathematical methods and statistical techniques presently available for hazard characterisation. The state of the art of mathematical modelling and quantitative methods used currently for regulatory decision-making in Europe and additional potential methods for risk assessment of chemicals in food and diet are described. Existing practices of JECFA, FDA, EPA, etc., are examined for their similarities and differences. A framework is established for the development of new and improved quantitative methodologies. Areas for refinement, improvement and increase of efficiency of each method are identified in a gap analysis. Based on this critical evaluation, needs for future research are defined. It is concluded from our work that mathematical modelling of the dose-response relationship would improve the risk assessment process. An adequate characterisation of the dose-response relationship by mathematical modelling clearly requires the use of a sufficient number of dose groups to achieve a range of different response levels. This need not necessarily lead to an increase in the total number of animals in the study if an appropriate design is used. Chemical-specific data relating to the mode or mechanism of action and/or the toxicokinetics of the chemical should be used for dose-response characterisation whenever possible. It is concluded that a single method of hazard characterisation would not be suitable for all kinds of risk assessments, and that a range of different approaches is necessary so that the method used is the most appropriate for the data available and for the risk characterisation issue. Future refinements to dose-response characterisation should incorporate more clearly the extent of uncertainty and variability in the resulting output.

Methods to identify and characterize developmental neurotoxicity for human health risk assessment. I: behavioral effects.

Alterations in nervous system function after exposure to a developmental neurotoxicant may be identified and characterized using neurobehavioral methods. A number of methods can evaluate alterations in sensory, motor, and cognitive functions in laboratory animals exposed to toxicants during nervous system development. Fundamental issues underlying proper use and interpretation of these methods include a) consideration of the scientific goal in experimental design, b) selection of an appropriate animal model, c) expertise of the investigator, d) adequate statistical analysis, and e) proper data interpretation. Strengths and weaknesses of the assessment methods include sensitivity, selectivity, practicality, and variability. Research could improve current behavioral methods by providing a better understanding of the relationship between alterations in motor function and changes in the underlying structure of these systems. Research is also needed to develop simple and sensitive assays for use in screening assessments of sensory and cognitive function. Assessment methods are being developed to examine other nervous system functions, including social behavior, autonomic processes, and biologic rhythms. Social behaviors are modified by many classes of developmental neurotoxicants and hormonally active compounds that may act either through neuroendocrine mechanisms or by directly influencing brain morphology or neurochemistry. Autonomic and thermoregulatory functions have been the province of physiologists and neurobiologists rather than toxicologists, but this may change as developmental neurotoxicology progresses and toxicologists apply techniques developed by other disciplines to examine changes in function after toxicant exposure.

Methods to identify and characterize developmental neurotoxicity for human health risk assessment. III: pharmacokinetic and pharmacodynamic considerations.

We review pharmacokinetic and pharmacodynamic factors that should be considered in the design and interpretation of developmental neurotoxicity studies. Toxicologic effects on the developing nervous system depend on the delivered dose, exposure duration, and developmental stage at which exposure occurred. Several pharmacokinetic processes (absorption, distribution, metabolism, and excretion) govern chemical disposition within the dam and the nervous system of the offspring. In addition, unique physical features such as the presence or absence of a placental barrier and the gradual development of the blood--brain barrier influence chemical disposition and thus modulate developmental neurotoxicity. Neonatal exposure may depend on maternal pharmacokinetic processes and transfer of the xenobiotic through the milk, although direct exposure may occur through other routes (e.g., inhalation). Measurement of the xenobiotic in milk and evaluation of biomarkers of exposure or effect following exposure can confirm or characterize neonatal exposure. Physiologically based pharmacokinetic and pharmacodynamic models that incorporate these and other determinants can estimate tissue dose and biologic response following in utero or neonatal exposure. These models can characterize dose--response relationships and improve extrapolation of results from animal studies to humans. In addition, pharmacologic data allow an experimenter to determine whether exposure to the test chemical is adequate, whether exposure occurs during critical periods of nervous system development, whether route and duration of exposure are appropriate, and whether developmental neurotoxicity can be differentiated from direct actions of the xenobiotic.

Common mechanism of toxicity: a case study of organophosphorus pesticides.

The Food Quality Protection Act of 1996 (FQPA) requires the EPA to consider "available information concerning the cumulative effects of such residues and other substances that have a common mechanism of toxicity ... in establishing, modifying, leaving in effect, or revoking a tolerance for a pesticide chemical residue." This directive raises a number of scientific questions to be answered before the FQPA can be implemented. Among these questions is: What constitutes a common mechanism of toxicity? The ILSI Risk Science Institute (RSI) convened a group of experts to examine this and other scientific questions using the organophosphorus (OP) pesticides as the case study. OP pesticides share some characteristics attributed to compounds that act by a common mechanism, but produce a variety of clinical signs of toxicity not identical for all OP pesticides. The Working Group generated a testable hypothesis, anticholinesterase OP pesticides act by a common mechanism of toxicity, and generated alternative hypotheses that, if true, would cause rejection of the initial hypothesis and provide criteria for subgrouping OP compounds. Some of the alternative hypotheses were rejected outright and the rest were not supported by adequate data. The Working Group concluded that OP pesticides act by a common mechanism of toxicity if they inhibit acetylcholinesterase by phosphorylation and elicit any spectrum of cholinergic effects. An approach similar to that developed for OP pesticides could be used to determine if other classes or groups of pesticides that share structural and toxicological characteristics act by a common mechanism of toxicity or by distinct mechanisms.

A rapid and efficient method for the radiosynthesis and purification of [125I]SCH23982.

The radiosynthesis of (1R)-(+)-1-phenyl-3-methyl-7-[125I]iodo-8-hydroxy- 2,3,4,5-tetrahydro-1H-3-benzazepine (commonly referred to as SCH23982) and its use as a high affinity D1 dopamine antagonist ligand have been reported previously. We now provide a simple and inexpensive protocol for the rapid and efficient synthesis of this radioligand based on the Cloramine-T-catalyzed reaction between the commercially available precursor (R)-(+)-1-phenyl-3-methyl- 8-hydroxy-2,3,4,5-tetrahydro-1H-3-benzazepine and carrier-free sodium [125I]iodide. [125I]SCH23982 is separated rapidly (within 20 min) from the precursor and reaction byproducts (e.g., chlorinated precursor, SCH23390) by reverse-phase HPLC on a C-8 column. The major iodinated product has been identified as SCH23982 based on co-chromatography with authentic SCH23982, UV spectral characteristics, and biological activity. The chromatographic effluent containing the active product is adsorbed on a C-18 Sep-Pak cartridge to remove mobile-phase constituents and permit it to be eluted and diluted to the desired concentration; this technique is used also for periodic repurification. Our synthesis protocol results in final purified product that incorporates ca. 50% of the initial 125I (tested using starting quantities of 1-10 mCi Na125I); the final product has a specific activity of ca. 2500 +/- 350 Ci/mmol. Data from in vitro receptor autoradiographic and homogenate studies with this radioligand are consistent with previously reported values in terms of expected receptor distribution, affinity, and density (KD of 1.0 nM, Bmax of 1400 fmol/mg protein in rat striatal membranes).

Alterations in the gamma-aminobutyric acid-gated chloride channel following transient forebrain ischemia in the gerbil.

The role of inhibitory neurotransmission in selective neuronal degeneration after transient forebrain ischemia was studied by binding of t-[35S]butylbicyclophosphorothionate ([35S]TBPS) to the gamma-aminobutyric acid (GABA)-gated chloride channel and measurement of GABAA receptor function in Mongolian gerbil brain. [35S]TBPS binding to the hippocampus, striatum, and cortex quantified by autoradiography and muscimol-stimulated 36Cl- uptake in synaptoneurosomes of the same regions were examined 1, 4, and 29 days after a 5-min bilateral carotid occlusion. [35S]TBPS binding was decreased in the pyramidal cell dendritic layers, stratum oriens, and stratum lacunosum-moleculare of the CA1 hippocampus, 4 and 29 days after occlusion, and in the stratum radiatum 29 days after occlusion. [35S]TBPS binding sites in the lateral striatum decreased 47% 4 days after occlusion. At the same time, there was a corresponding decrease in muscimol-stimulated 36Cl- uptake in the striatal synaptoneurosomes. Muscimol-stimulated 36Cl- uptake in the hippocampus decreased slightly 4 days after occlusion and more so after 29 days, although these decreases were not significant. No changes were observed in somatosensory cortex at any time point. These data suggest that a portion of GABAA receptors in areas sensitive to ischemic insult are associated with degenerating neurons, whereas other GABAA) receptors are spared.

Cellular regulation of the benzodiazepine/GABA receptor: arachidonic acid, calcium, and cerebral ischemia.

The effects of cellular mediators that contribute to ischemia-induced neuronal degeneration on gamma-aminobutyric acid (GABAA)-receptor function were studied. In vitro, phospholipase A2 (PLA2) inhibited muscimol-induced 36Cl- uptake in cerebral cortical synaptoneurosomes. The major hydrolysis product of PLA2 activity, arachidonic acid, also inhibited GABA-mediated 36Cl- uptake. The unsaturated nature of arachidonic acid makes it (and its metabolites) highly susceptible to peroxidation by oxygen radicals. Incubation of synaptoneurosomes with the superoxide radical-generating system, xanthine and xanthine oxidase, decreased muscimol-induced 36Cl- uptake, suggesting that the peroxidation of arachidonic acid and/or its metabolites interferes with GABAA-receptor function. Another factor involved in ischemia-induced neuronal degeneration is an increase in intracellular Ca2+. Calcium also inhibited GABA-mediated 36Cl- flux, consistent with its ability to activate PLA2. In contrast, Mg2+, which blocks Ca2+ channels, enhanced muscimol-induced 36Cl- uptake, consistent with its neuroprotective effects. Each of these cellular processes is activated during cerebral ischemia and can lead to neuronal degeneration. We used a model of transient forebrain ischemia in gerbils to determine if GABAA-receptor regulation is altered in vivo at a time when CA1 hippocampal cells have degenerated. Four days after a 5 minute bilateral carotid artery occlusion, receptor autoradiography was performed to measure the binding of [35S]t-butylbicyclophosphorothionate (TBPS) to the GABA-gated chloride channel. Significant decreases in TBPS binding were observed only in the dendritic layers (stratum oriens and lacunosem moleculare) of the CA1 hippocampus. The results suggest that ischemia-induced cellular processes that contribute to cell death can decrease GABA-gated chloride channels on dendrites of CA1 pyramidal cells, and that GABAA receptors may also reside on neurons afferent to or intrinsic to the dendritic layers of CA1 hippocampus.

Dopamine receptor 'supersensitivity' occurring without receptor up-regulation.

Unilateral 6-hydroxydopamine (6-OHDA)-induced lesions of the substantia nigra have been widely used to study various aspects of dopamine neurobiology, and to screen for antiparkinsonian drugs. This study examined the role of receptor alterations in the pharmacological supersensitivity seen in response to lesioning of central dopamine pathways in rats by intracisternal (IC) administration of 6-OHDA (200 micrograms), as well as by bilateral (BIL) or unilateral (UNI) infusion of 6-OHDA into the substantia nigra (8 micrograms/side). Both IC and BIL lesions resulted in permanent decreases in dopamine concentration in the striatum, the major terminal projection from the substantia nigra. When challenged with apomorphine (0.3 mg/kg), IC-lesioned rats exhibited bursts of rapid locomotion interspersed by rearing, whereas BIL-lesioned rats displayed intense grooming or gnawing and nose poking of the cage floor; these behaviors were not seen in respective sham (i.e. vehicle)-lesioned rats injected with apomorphine. Scatchard analysis of saturation isotherms of both D1 [( 3H]SCH23390 binding sites) and D2 [( 3H]spiperone binding sites) dopamine receptors in the striatum revealed no difference in either the maximum number of binding sites (Bmax), or the dissociation constant (Kd) of either receptor type when BIL and IC lesioned rats were compared to appropriate controls. Conversely, the UNI lesioned rats had, under identical conditions of analysis, the expected increase in the density of D2 receptors on the lesioned side. There was no change in dopamine-sensitive adenylate cyclase activity in the striata of supersensitive IC-lesioned rats, but there was a shift to the left in the dose-response curve in striata from rats bilaterally-lesioned in the substantia nigra, similar to what occurs in UNI lesioned rats. Together, these data clearly demonstrate that although increases in receptor density and changes in cAMP systems are seen in the UNI model, neither mechanism is a requirement for functional supersensitivity in response to 6-OHDA lesions. These data suggest that other cellular events (e.g. alterations in receptor interactions) may play a role in the response to insult, and raise questions about the utility of the unilateral model as a screen for antiparkinsonian drugs.

The use of locomotor activity as a behavioral screen for neuronal damage following transient forebrain ischemia in gerbils.

Five min bilateral carotid artery occlusion (BCO) in gerbils results in selective degeneration of neurons in the hippocampus, striatum and cortex, and an increase in spontaneous locomotor activity. These phenomena were examined to determine if an association could be made between the site or degree of neuronal degeneration and the increase in locomotor activity. The distance traveled by the BCO gerbils in a novel cage 1, 4, and 28 days after a 5 min occlusion was significantly greater than control. The extensive pyramidal cell damage in the CA1 region of the hippocampus in BCO gerbils was associated with the significant increase in locomotor activity. The increase in locomotor activity did not correlate with either the striatal or cortical damage present. The increase in gerbil locomotor activity following a 5 min BCO can be used as a predictor of CA1 damage, but not as a predictor of striatal or cortical damage.