Stephen M. Lasley

The speciation of metals in mammals influences their toxicokinetics and toxicodynamics and therefore human health risk assessment.

Chemical form (i.e., species) can influence metal toxicokinetics and toxicodynamics and should be considered to improve human health risk assessment. Factors that influence metal speciation (and examples) include: (1) carrier-mediated processes for specific metal species (arsenic, chromium, lead and manganese), (2) valence state (arsenic, chromium, manganese and mercury), (3) particle size (lead and manganese), (4) the nature of metal binding ligands (aluminum, arsenic, chromium, lead, and manganese), (5) whether the metal is an organic versus inorganic species (arsenic, lead, and mercury), and (6) biotransformation of metal species (aluminum, arsenic, chromium, lead, manganese and mercury). The influence of speciation on metal toxicokinetics and toxicodynamics in mammals, and therefore the adverse effects of metals, is reviewed to illustrate how the physicochemical characteristics of metals and their handling in the body (toxicokinetics) can influence toxicity (toxicodynamics). Generalizing from mercury, arsenic, lead, aluminum, chromium, and manganese, it is clear that metal speciation influences mammalian toxicity. Methods used in aquatic toxicology to predict the interaction among metal speciation, uptake, and toxicity are evaluated. A classification system is presented to show that the chemical nature of the metal can predict metal ion toxicokinetics and toxicodynamics. Essential metals, such as iron, are considered. These metals produce low oral toxicity under most exposure conditions but become toxic when biological processes that utilize or transport them are overwhelmed, or bypassed. Risk assessments for essential and nonessential metals should consider toxicokinetic and toxicodynamic factors in setting exposure standards. Because speciation can influence a metals fate and toxicity, different exposure standards should be established for different metal species. Many examples are provided which consider metal essentiality and toxicity and that illustrate how consideration of metal speciation can improve the risk assessment process. More examples are available at a website established as a repository for summaries of the literature on how the speciation of metals affects their toxicokinetics. This article is based on a workshop entitled “Metal Speciation in Toxicology: Determination and Importance for Risk Assessment” presented at the 42nd Annual Meeting of the Society of Toxicology, March, 2003, Salt Lake City, UT.

Presynaptic glutamatergic function in dentate gyrus in vivo is diminished by chronic exposure to inorganic lead.

Reductions in membrane Ca2+ channel currents and depolarization-evoked neurotransmitter release have been repeatedly observed as a result of acute exposure to Pb2+. This study was performed to determine whether hippocampal glutamate and GABA release are impaired in intact animals chronically exposed to lead (Pb). As paired-pulse facilitation in the hippocampus is primarily mediated by an enhancement of glutamate release, this neurophysiological measure was also assessed in the dentate gyrus of Pb-exposed animals. Pregnant dams received 0.2% Pb acetate in the drinking water at parturition, and male offspring were weaned to the same solution as that given their dams. Control animals were maintained on distilled water. As adults, animals had intracerebral dialysis probes inserted through guide cannulae implanted 2-4 days previously and the hippocampal CAI-dentate area was perfused with modified Ringers solution. Transmitter release was induced by perfusion with 150 mM K+ with half the animals in each group tested with Ca2+ present in the perfusate (total release) and the other half with Ca2+ absent (Ca(2+)-independent release). K(+)-stimulated total glutamate release was reduced in Pb-exposed animals relative to controls. No group differences were observed under Ca(2+)-free conditions, indicating that Ca(2+)-dependent glutamate release was decreased in exposed rats. In contrast no group differences in K(+)-stimulated total GABA release were evident, whereas an augmentation in GABA release under Ca(2+)-free conditions was revealed in Pb-exposed animals. The effects of exposure on the Ca(2+)-dependent components of release are consistent with in vitro evidence indicating an inhibitory action of Pb2+ at voltage-sensitive Ca2+ channels. A separate group of animals was prepared under urethane anesthesia with stimulating and recording electrodes placed in the perforant path and dentate gyrus, respectively. Pairs of stimulus pulses were delivered at interpulse intervals (IPI) of 10-250 ms. Pb exposure induced an increase in paired-pulse depression at the 20 ms 1PI and reduced paired-pulse facilitation at the 30 ms IPI. Decreases in paired-pulse facilitation could not be attributed to the reported effects of Pb2+ on N-methyl-D-aspartate (NMDA) receptors as MK-801 (1.0 mg/kg, s.c.) administration produced an opposing pattern of effects on paired-pulse measures. The Pb-induced suppression of paired-pulse facilitation is consistent with exposure-related decreases in total glutamate release. The impact of these effects of Pb exposure on hippocampal glutamatergic transmission may contribute to the reported effects of Pb on other forms of synaptic plasticity including long-term potentiation, a model of learning and memory.

CHRONIC EXPOSURE TO ENVIRONMENTAL LEVELS OF LEAD IMPAIRS IN VIVO INDUCTION OF LONG-TERM POTENTIATION IN RAT HIPPOCAMPAL DENTATE

This study examined the effects of chronic developmental lead (Pb) exposure in rats on hippocampal long-term potentiation (LTP). Male offspring were exposed to 0.2% Pb acetate continuously from birth until testing at 85-105 days. Excitatory postsynaptic potential (EPSP) and population spike amplitudes were measured in the dentate hilar region in response to stimulation applied to the lateral perforant path. LTP was induced in control animals with an average maximal EPSP potentiation of 41%, which was significantly greater than the increase in EPSP amplitudes (2%) in exposed animals after tetanizing stimulation. Current-voltage curves in controls demonstrated significant increases in EPSPs and population spikes after application of pulse trains to induce LTP, while exposed rats exhibited no discernible change in responses. These findings suggest that induction or development of LTP in the dentate hilar region in vivo is impaired by chronic developmental exposure to environmentally relevant levels of Pb.

Roles of neurotransmitter amino acids in seizure severity and experience in the genetically epilepsy-prone rat

This investigation was designed to compare seizure-naive and seizure-experienced genetically epilepsy-prone rats (GEPRs) in order to distinguish transmitter amino acid changes related to seizure severity from those associated with seizure experience. Moderate (GEPR-3) and severe (GEPR-9) seizure male GEPRs were divided into seizure-naive and seizure-experienced groups based on whether seizure-inducing acoustical stimuli had been presented between 45 and 60 days of age, and then were sacrificed at 76 +/- 3 days. gamma-Aminobutyric acid (GABA) concentrations were lower in both GEPR-3s and GEPR-9s compared to non-epileptic controls in each brain region examined. Aspartate content was elevated in 5 of 6 brain areas in GEPR-9s compared to non-epileptic controls, and in 3 regions was higher in GEPR-9s than in GEPR-3s. In contrast, taurine concentrations were higher in GEPR-3s than in non-epileptic controls in each region, and in 4 areas were higher in GEPR-3s than in GEPR-9s. Changes resulting from seizure experience consisted of increases in aspartate, glutamate and glycine in seizure-experienced compared to seizure-naive groups in inferior colliculus and in motor-sensory and frontal cortices. These findings suggest that the high levels of taurine in GEPR-3s and the elevated content of aspartate in GEPR-9s have roles as determinants of seizure severity. The low concentrations of GABA in both types of GEPRs are consistent with a role for this amino acid in determination of seizure susceptibility. Furthermore, the seizure-induced changes in aspartate and glutamate in both types of GEPRs support the concept that these excitatory amino acids mediate changes in seizure predisposition.(ABSTRACT TRUNCATED AT 250 WORDS)

Rat hippocampal NMDA receptor binding as a function of chronic lead exposure level.

Chronic developmental lead (Pb) exposure is known to impair cognitive ability in children and young animals. These findings have led to research examining exposure effects on long-term potentiation (LTP), a model of synaptic plasticity, and on NMDA receptor function. This study determined the changes occurring in hippocampal 3H-MK-801 binding as a function of exposure level for comparison to changes in LTP previously reported from this laboratory. Dams were exposed to 0.1%, 0.2%, 0.5% and 1.0% Pb in the drinking water beginning at parturition, and male offspring were weaned to the same solutions as their dams and maintained on these regimens until assessment as adults. A crude membrane fraction was prepared from hippocampal tissue, and Scatchard analysis conducted in the presence of saturating concentrations of glutamate and glycine. NMDA receptor density was elevated as a result of Pb exposure with significant increases in the 0.2% (38%) and 0.5% (30%) groups compared to control group values. No changes were observed in the 0.1% and 1.0% animals, thus constituting a biphasic dose-effect relationship. These findings are an approximate reflection of analogous relationships reported for hippocampal LTP and glutamate release, suggesting that the diminished glutamate release is one cause of the receptor up-regulation. However, since increases in receptor number were uncovered, it is unlikely that changes in NMDA receptor density constitute a primary mechanism whereby Pb impairs hippocampal LTP.

Diminished regulation of mesolimbic dopaminergic activity in rat after chronic inorganic lead exposure

Previous studies in this laboratory have indicated that chronic lead (Pb) exposure during development induces a neurotoxicity in dopamine (DA) neurons that is primarily presynaptic in nature and at least partially related to altered regulation of DA synthesis. A primary form of DA synthesis regulation is the inhibition exerted on synaptic tyrosine hydroxylase activity via dopaminergic autoreceptors. This study assessed the functional status of this mechanism in Pb-exposed rats employing a pharmacological model. At parturition dams received 0.2% Pb acetate (1090 ppm) in the drinking water while control dams received distilled water. Offspring were weaned to and maintained on the same solution given their dams until termination at 125 days. Rats were given saline or 6,7-dihydroxy-2-dimethylaminotetralin (TL-99, 2.5-20 mg/kg ip) 40 min before termination followed 10 min later by 750 mg/kg ip of gamma-butyrolactone (GBL) or saline. The ability of TL-99 to prevent the GBL-induced increase in DA content was significantly diminished in nucleus accumbens (NAc) of exposed rats compared to controls, indicating that chronic Pb impairs receptor-mediated regulation of DA synthesis in mesolimbic neurons. No effect of Pb was observed in caudate-putamen. In animals receiving only saline injections concentrations of the DA metabolites, homovanillic acid and dihydroxyphenylacetic acid, were significantly decreased by Pb in the range of 17-31% and 12-24%, respectively. DA content was also significantly diminished by Pb in ventral tegmental area of these latter groups. These findings suggest that chronic Pb has multiple actions on central nervous system dopaminergic neurons consisting of an impaired regulation of DA synthesis that is apparently independent of a decrease in DA release.

Developmental thyroid hormone insufficiency reduces expression of brain-derived neurotrophic factor (BDNF) in adults but not in neonates.

Brain-derived neurotrophic factor (BDNF) is a neurotrophin critical for many developmental and physiological aspects of CNS function. Severe hypothyroidism in the early neonatal period results in developmental and cognitive impairments and reductions in mRNA and protein expression of BDNF in a number of brain regions. The present study examined the impact of modest levels of developmental thyroid hormone insufficiency on BDNF protein expression in the hippocampus, cortex and cerebellum in the neonatal and adult offspring of rat dams treated throughout pregnancy and lactation. Graded levels of hormone insufficiency were induced by adding propylthiouracil (PTU, 0, 1, 2, 3 and 10 ppm) to the drinking water of pregnant dams from early gestation (gestational day 6) until weaning of the pups. Pups were sacrificed on postnatal days (PN) 14 and 21, and -PN100, and trunk blood collected for thyroid hormone analysis. Hippocampus, cortex, and cerebellum were separated from dissected brains and assessed for BDNF protein. Dose-dependent reductions in serum hormones in dams and pups were produced by PTU. Consistent with previous findings, age and regional differences in BDNF concentrations were observed. However, no differences in BDNF expression were detected in the preweanling animals as a function of PTU exposure; yet dose-dependent alterations emerged in adulthood despite the return of thyroid hormone levels to control values. Males were more affected by PTU than females, BDNF levels in hippocampus and cortex were altered but not those in cerebellum, and biphasic dose-response functions were detected in both sexes. These findings indicate that BDNF may mediate some of the adverse effects accompanying developmental thyroid hormone insufficiency, and reflect the potential for delayed impact of modest reductions in thyroid hormones during critical periods of brain development on a protein important for normal synaptic function.

Amino acids, monoamines and audiogenic seizures in genetically epilepsy-prone rats: effects of aspartame

It has been suggested that aspartame facilitates seizures in man and animals because phenylalanine, one of its major metabolites, interferes with brain transport of neurotransmitter precursors and alters the synthesis of monoamine neurotransmitters such as norepinephrine, dopamine and/or serotonin. This facilitation is purportedly more likely in subjects predisposed to seizures. One test of this hypothesis would be to administer a wide range of aspartame doses to subjects whose seizure predisposition is dependent on abnormalities in monoaminergic function. Genetically epilepsy-prone rats (GEPRs) have a broadly based seizure predisposition that is based, in part, on widespread central nervous system noradrenergic and serotonergic deficits. Further reductions in the functional state of these neurotransmitters increases seizure severity in GEPRs. Thus, GEPRs appear ideally suited for testing the hypothesis that aspartame facilitates seizures by interfering with central nervous system monoamines. Oral administration of acute (50-2000 mg/kg) or sub-chronic (up to 863 mg/kg/day for 28 days) doses of aspartame did not alter seizure severity in either of two types of GEPRs. Not surprisingly, acute aspartame doses produced dramatic changes in plasma and brain amino acid concentrations. Hypothesized alterations in monoamine neurotransmitter systems were largely absent. Indeed, increases in norepinephrine concentration, rather than the hypothesized decreases, were the most evident alterations in these neurotransmitter systems. We conclude that aspartame does not facilitate seizures in GEPRs and that convincing evidence of seizure facilitation in any species is lacking.

Diminished potassium-stimulated GABA release in vivo in genetically epilepsy-prone rats

This experiment was conducted to assess the physiological relevance of observed changes in transmitter amino acid content in severe seizure genetically epilepsy-prone rats (GEPR-9s) by use of microdialysis. Adult male GEPR-9s and non-epileptic control rats were implanted with guide cannulae, and 6 mm (loop) dialysis probes were inserted unilaterally into rostral caudate and perfused with artificial cerebrospinal fluid. Each subject was perfused in the awake state with 100 or 150 mM K+ for 80 min in separate counterbalanced sessions, and 20-min fractions collected. High K+ perfusion increased extracellular fluid GABA and glutamate (GLU) in a concentration-dependent manner in both GEPR-9s and non-epileptic control rats. However, in the presence of 150 mM K+ GABA release was decreased in GEPR-9s relative to controls throughout the stimulation interval. In contrast, the increase in extracellular fluid GLU after high K+ was not different in the two groups. These results suggest an important role for mechanisms underlying GABA release in the seizure susceptibility observed in GEPR-9s.

The combined EEG-intracerebral microdialysis technique: a new tool for neuropharmacological studies on freely behaving animals

In this study we combined EEG and intracerebral microdialysis techniques in freely behaving rats. Various drugs were delivered into the hippocampus and cerebral cortex by means of microdialysis and, simultaneously, the EEG activity of the dialyzed area was monitored. The microdialysis procedure itself, when artificial cerebrospinal fluid was perfused, did not change the normal hippocampal or cortical EEG pattern. Drug inclusions into the microdialysis fluid, however, caused marked changes in the electrical activity of the dialyzed sites. In this report we present the following examples: (1) the dose-dependent spike-provoking effect of NMDA in hippocampus, (2) the potentiation of this NMDA effect in hippocampus by dibutyryl cyclic AMP, and (3) the EEG depressant effect of high concentration of K+ in the cerebral cortex. The artificial cerebrospinal fluid and drug solutions were alternated in the microdialysis system with a 2-way valve placed outside the test chamber. As a consequence, the drugs were delivered into the brain without interrupting the ongoing behavior, including sleep, of the examined animals. This study shows that the combined EEG-intracerebral microdialysis technique is a useful tool, with many unique advantages, for in vivo neuropharmacological studies.