An assessment of neurotoxicity of aroclors 1016, 1242, 1254, and 1260 administered in diet to Sprague-Dawley rats for one year.

As part of a comparative chronic toxicity/oncogenicity study of different Aroclors (1016, 1242, 1254, and 1260), neurotoxicity was assessed in male and female Sprague-Dawley rats using functional observational battery (FOB) and motor activity tests, and histopathologic evaluation of selected nervous system tissues. Doses varied by Aroclor and ranged from 25 to 200 ppm in the diet. Animals were evaluated prior to initiation of dosing and at 13, 26, 39, and 52 weeks of exposure. Clinical signs, body weights, and feed consumption were evaluated weekly. Data analysis of FOB and motor activity results revealed several instances where Aroclor-treated groups were different from control. However, these were considered incidental, as they lacked any consistent dose- or time-related pattern that would suggest Aroclor-induced neurotoxicity. The nonremarkable findings during each of the four assessments were supported by the absence of any treatment-related clinical signs or mortality. Decreased body weight gain was evident in the male 100 ppm Aroclor 1254 dose group and in all female Aroclor 1254 dose groups late in the study (when a linear relationship was assumed between body weight and time), correlating with decreased feed consumption. Although a variety of incidental, spontaneous, degenerative changes were found in nervous tissue evaluated histopathologically, these changes were seen with similar incidence and severity in treated and control groups. No lesions were found that could be attributed to Aroclor-related neurotoxicity. In summary, 52 weeks of exposure to Aroclors 1016, 1242, 1254, or 1260 mixed in the diet did not yield any functional or morphologic changes indicative of PCB-induced neurotoxicity.

Comparative absorption of lead from contaminated soil and lead salts by weanling Fischer 344 rats.

A 44-day dosed feed study was performed to compare the bioavailability of lead from contaminated soil versus two lead salts and the effect of soil on gastrointestinal absorption of ingested lead. Male Fischer rats (approximately 4 weeks of age) received lead, 17, 42, or 127 ppm, in the form of lead acetate, lead sulfide, lead-contaminated soil, or combinations thereof in the diet for 7, 15, or 44 days. Control soil was added to the diets of some animals to determine how it might alter lead bioavailability. Blood Delta-aminolevulinic acid dehydratase (Delta-ALAD) and blood, bone, kidney, and liver lead were determined in groups of animals at each time-point. Blood Delta-ALAD was inhibited in a dose-dependent manner and to the greatest degree in the lead acetate and lead acetate/control soil groups, followed by the lead sulfide and lead-contaminated soil groups. Bone and tissue lead levels increased in a dose-dependent manner and were greatest in animals receiving lead acetate and significantly less in animals receiving lead sulfide and lead-contaminated soil. Blood lead levels were generally greatest by 7 days and stabilized at lower levels thereafter. Bone lead concentration-time patterns did not demonstrate the biphasic change seen with tissues and continued to increase in most treatment groups through the course of the study. The presence of soil in the diet clearly attenuated the absorption of lead acetate, but had little effect on the absorption of lead sulfide. Results of these studies confirm previous observations that lead absorption is highly dependent on the form of lead ingested and the matrix in which it is ingested. More important, these studies demonstrate that lead in soil may be significantly less available than estimated by current default assumptions and that the presence of soil may decrease the availability of lead from lead salts on which the default assumptions are based. Results presented here also demonstrate that the weanling rat may represent an appropriate model that could be used to obtain relatively rapid and economical estimates of the availability of lead in complex matrices such as soil.

Evaluating lead bioavailability data by means of a physiologically based lead kinetic model.

A method of bioavailability estimation is presented in which a physiologically based kinetic model of lead kinetics is fit simultaneously to blood and bone lead concentrations after a period of exposure to dietary lead. Optimization of the simultaneous fit, varying only fractional absorption, gives the best estimate of fractional bioavailability for each treatment group. The analysis was applied to data from three separate studies in which rats were fed for 30 consecutive days purified diets containing lead added as lead acetate, mine waste-contaminated test soils, or mine waste itself. Fractional absorption decreased as lead intake increased, regardless of the source of the lead; but the magnitude of this dose dependence was lead source-dependent. There were no differences in lead absorption by male and female rats when lead intake was expressed per unit body weight. Fractional absorption varied from 4 to 5%, at low exposure rates (1-2 mg lead/kg/day) when lead acetate was added to the diet, to 0.24% at a high exposure rate (24 mg/kg/day) when a mine waste-contaminated test soil was added to the diet. Comparison of the results of this analysis with the results of a more conventional analysis, in which the bone and blood lead concentrations were separately compared with bone and blood lead concentrations in rats given daily injections of lead acetate intravenously for 29 consecutive days, demonstrated that the standard analysis failed to reveal the dose dependence of fractional absorption.

Bioavailability of arsenic in soil and house dust impacted by smelter activities following oral administration in cynomolgus monkeys.

This study was conducted to determine the extent of arsenic (As) absorption from soil and house dust impacted by smelter activities near Anaconda, Montana. Female cynomolgus monkeys were given a single oral administration via gelatin capsules of soil (0.62 mg As/kg body wt) or house dust (0.26 mg As/kg body wt), or soluble sodium arsenate by the gavage or intravenous route of administration (0.62 mg As/kg body wt) in a crossover design with a minimum washout period of 14 days. Urine, feces, and cage rinse were collected at 24-hr intervals for 168 hr. Blood was collected at specified time points and area under the curves (AUCs) was determined. Arsenic concentrations for the first 120 hr, representing elimination of greater than 94% of the total administered dose for the three oral treatment groups, were < 0.021 to 4.68 micrograms/ml for the urine and < 0.24 to 31.1 micrograms/g for the feces. In general, peak concentrations of As in the urine and feces were obtained during the collection intervals of 0-24 and 24-72 hr, respectively. The main pathway for excretion of As for the intravenous and gavage groups was in the urine, whereas for the soil and dust groups, it was in the feces. Mean absolute percentage bioavailability values based on urinary excretion data were 68, 19, and 14% for the gavage, house dust, and soil treatments, respectively, after normalization of the intravenous As recovery data to 100%. Corresponding absolute bioavailability values based on blood were 91, 10, and 11%. The bioavailability of soil and house dust As relative to soluble As (by gavage) was between 10 and 30%, depending upon whether urinary or blood values were used. These findings suggest that risks associated with the ingestion of As in soil or dust will be reduced compared to ingestion of comparable quantities of As in drinking water.

Absolute bioavailability of lead acetate and mining waste lead in rats.

The primary purpose of this study was to generate data that could be used to determine the absolute bioavailability of lead using data from a previous study in which soil containing lead from mining waste was mixed with feed. Young male and female Sprague-Dawley rats (7-8 weeks of age, five/sex/group) were given either soluble lead acetate mixed in a purified diet (AIN-76) at three different dose levels (1, 25, and 250 ppm Pb for 30 consecutive days) or intravenously at doses of 0.02, 0.20, and 2.0 mg Pb/kg BW for 29 days. A control group (purified diet only) was also included. The intravenous groups were used to provide maximal absorption (lead presumed to be 100% bioavailable) and accumulation data for lead in blood, bone, and liver. The lead acetate groups were used to evaluate the comparability of the present study with a previous study that compared bioavailable lead from ingested soil and lead acetate. Group mean whole blood, bone and liver lead concentration values increased with increasing dose levels for all treatment groups. A linear relationship was observed between blood lead concentration and dose following intravenous administration of lead and this provided empirical support for using blood lead concentrations at supposed steady state (approximately 30 days) to compute the bioavailability of lead administered by different routes and from different sources. The absolute bioavailability values of mining waste lead in soil were low based on the results for all tissue types. Absolute bioavailability values for lead acetate in dosed feed for blood, bone, and liver were approximately 6-, 19-, and 20-fold greater, respectively, than mining waste lead. Based on the current design and test system used, the absolute bioavailability of mining waste lead in soil administered in feed was approximately 3% based on blood data and less than 1% based on bone and liver data. These data are consistent with the low solubility of the constituent lead mineral phases in Butte soils.

Bioavailability of arsenic in soil impacted by smelter activities following oral administration in rabbits.

This study determined the extent of arsenic (As) absorption from soil from Anaconda, Montana. Prepubescent male and female SPF New Zealand White rabbits (5/sex/group) were given a single oral (capsule) administration of soil (3900 ppm As) at three different dose levels (0.2, 0.5, and 1.0 g of soil/kg, corresponding to 0.78, 1.95, and 3.9 mg As/kg, respectively). Standard groups included untreated controls, an intravenous sodium arsenate group (1.95 mg As/kg), and a gavage sodium arsenate group (1.95 mg As/kg). Urine, cage rinse, and feces were collected at 24-hr intervals for 5 days and were analyzed for total As concentration. Clinical signs, body weights, and food consumption for treated animals were similar to controls. Maximum As concentrations were obtained over the initial 24-hr collection interval. A dose-dependent delay in urinary As excretion, the major elimination pathway, was observed in the oral soil group compared to that in the gavage group. For the animals in the soil groups, approximately 80% of the administered As dose was eliminated in the feces compared to approximately 10 and 50% for the intravenous and oral gavage groups, respectively. The relative oral bioavailabilities (+/- SD) of As in the gavage and test soil groups based on comparison with excreta data from the intravenous group were approximately 50 +/- 5.7 and 24 +/- 3.2%, respectively (after normalization of intravenous group's As recovery data to 100%). These results indicated that As in the soil was probably in a less soluble and therefore a less absorbable form than sodium arsenate.

Relative bioavailability of lead from mining waste soil in rats.

The purposes of this study were to determine the extent of absorption of lead (Pb) in mining waste soil from Butte, Montana, and to investigate the effect of mining waste soil dose (g soil/day) on tissue lead concentrations. Young, 7- to 8-week-old male and female Sprague-Dawley rats (5/sex/group) were given mining waste soil that contained 810 or 3908 ppm lead mixed in a purified diet (AIN-76) at four different dose levels (0.2, 0.5, 2, and 5% dietary soil) for 30 consecutive days. Standard groups included untreated controls and dosed feed soluble lead acetate groups (1, 10, 25, 100, and 250 micrograms Pb/g feed). The test soil dose levels bracketed a pica child's soil exposure level and the lead acetate concentrations bracketed the test soil dose levels of lead. Liver, blood, and femur were analyzed for total lead concentration using graphite furnace atomic absorption spectroscopy. Clinical signs, body weight, food consumption, and liver weights for test soil and standard groups were similar to control. Tissue lead concentrations from test soil animals were significantly lower than the tissue concentrations for the lead acetate group. Relative percentage bioavailability values, based on lead acetate as the standard, were independent of the two different test soils, dose levels, and sex and were only slightly dependent on the tissue (blood > bone, liver). Mean relative percentage bioavailability values of lead in the Butte mining waste soil were 20% based on the blood data, 9% based on the bone data, and 8% based on the liver data. The results of this study will provide the information needed to determine the significance of lead exposure from Butte soils in assessing human health risks as part of the Superfund Remedial Investigation/Feasibility Study process.

The cellular basis of delirium and its relevance to age-related disorders including Alzheimer's disease.

A wide variety of conditions lead to delirium (i.e., metabolic encephalopathies) in human beings and animals. Despite the varied etiology the clinical consequences are relatively stereotyped which suggests that the diverse insults that cause delirium may act by common metabolic and cellular "final pathways." Related molecular and cellular mechanisms may be involved in aging and Alzheimer's disease, conditions that predispose to the development of delirium. Animal models of delirium better reflect age-related disorders such as Alzheimer's disease than those that impair a single neurotransmitter system such as the cholinergic system; the metabolic encephalopathies produce global cognitive disturbance, which is more typical of these disorders. Thus, research related to delirium has far-reaching implications for normal and abnormal brain function.

Effects of dietary tyrosine on L-dopa- and amphetamine-induced changes in locomotor activity and neurochemistry in mice.

Recent findings suggest that intraperitoneal injections of L-tyrosine at high doses (100 mg/kg) alters amphetamine-induced changes in behavior by restoring amphetamine-induced decreases in whole brain norepinephrine (NE). The present study examined the motor effects of L-dihydroxyphenylalanine (L-dopa) and d-amphetamine sulfate in mice after treatment with a basal casein diet supplemented with L-tyrosine. The basal diet supplemented with 1-4% L-tyrosine, or 1-4% L-phenylalanine, produced no changes in motor activity in otherwise untreated mice. Whereas L-dopa (25-100 mg/kg) following inhibition of extracerebral decarboxylase by Ro 4-4602 (25 mg/kg) slightly decreased activity in diet control (casein) animals, this drug treatment enhanced motor activity in a dose-related fashion when L-tyrosine was added to the diet. Increases in motor activity following low doses of amphetamine (0.75-1.5 mg/kg) in casein control mice were antagonized by dietary L-tyrosine, but a higher dose of d-amphetamine (3 mg/kg) interacted with the addition of L-tyrosine producing an increase in motor activity. Neurochemical changes observed in brain concentrations of tyrosine, dopamine (DA), norepinephrine (NE), dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), tryptophan, serotonin (5-HT) and 5-hydroxy-indoleacetic acid (5-HIAA) following drug and diet treatments suggest that 5-HT systems, in addition to catecholamine systems, may be involved in mediating these effects.

Selective damage in striatum and hippocampus with in vitro anoxia.

An in vitro model of anoxia-induced brain damage was utilized to help elucidate the biochemical basis of cell damage due to reduced oxygen availability. Previous studies suggest that anoxia-induced damage may vary presynaptically, post-synaptically or in the cell body. Thus, the consequences of an anoxic treatment incubation were examined with hippocampal slices, which contain cholinergic nerve terminals but not cell bodies, and with slices from whole striatum or its subregions, which contain both cholinergic cell bodies and nerve terminals. Slices were preincubated with either oxygen or nitrogen (treatment incubation) and the persistent effects of this treatment on [14C]acetylcholine and 14CO2 production from [U-14C]glucose were assessed in a subsequent incubation under optimal conditions (test incubation). An anoxic treatment incubation reduced the subsequent test incubation production of CO2 about 40% in the hippocampus and striatum. The anoxic treatment incubation diminished ACh production by 46% in the striatum, but only minimally affected that in the hippocampus. Anoxic treatment incubations of synaptosomes did not alter test-incubation ACh synthesis or CO2 production. Omission of calcium from the anoxic treatment incubation increased striatal ACh synthesis by 88% and CO2 production in both regions. These results suggest that anoxia produces persistent changes in postsynaptic processes or cell bodies (in this model cholinergic ones) that differ from those in nerve terminals and that calcium is important in the production of these deficits.

Dopamine, acetylcholine, and glutamate interactions in aging. Behavioral and neurochemical correlates.

Aging, hypoxia, and thiamin deficiency diminish motor performance. Similar alterations of ACh, DA, and glutamate metabolism accompany hypoxia, thiamin deficiency, and aging. Both aging and hypoxia reduce ACh release and stimulate DA and glutamate release. Presynaptic enhancement of DA and glutamate release may be important in the production of cell damage that may contribute, in part, to age-related deficits in motor as well as cognitive function. The decline in ACh release may be important in the production of the cognitive deficits. An understanding of the interactions of neurotransmitters in hypoxia and thiamin deficiency aids our understanding of normal aging and increases the possibility of developing better treatments for the multiple neurotransmitter deficiencies that accompany many metabolic, age-related, and chronic degenerative disorders.

Differential alteration of dopamine, acetylcholine, and glutamate release during anoxia and/or 3,4-diaminopyridine treatment.

The potassium-stimulated release of acetylcholine (ACh), glutamate (GLU) and dopamine (DA) from mouse striatal slices was studied during anoxia and/or 3,4-diaminopyridine (DAP) treatment. Anoxia, in the presence of calcium, increased DA and GLU release, but depressed ACh release. Omission of calcium from an anoxic incubation further stimulated GLU and DA release and impaired ACh release. Under normoxic conditions, DAP (100 microM) increased the release of all three neurotransmitters; the sensitivity of the slices to DAP changed with the presence or absence of an acetylcholinesterase inhibitor in the preincubation media. During an anoxic incubation, DAP did not ameliorate the anoxic-induced, K+-stimulated impairment of ACh release, but significantly reduced the K+-stimulated release of GLU and DA. These results are consistent with the hypothesis that hypoxia induces a presynaptic deficit that may underlie postsynaptic ischemic-induced changes. Amelioration of these presynaptic alterations in neurotransmitter release may be an effective approach to preventing hypoxic-induced damage.

Effect of age on behavioral and enzymatic changes during thiamin deficiency.

Open field behavior and whole brain enzymatic activities were determined during thiamin deficiency in two strains of young, as well as in aged mice. In young CD-1 mice, thiamin deficiency reduced total distance traveled and vertical movements after 7 days and the decline was more than 50% by day 9. The behavioral deficit was highly correlated to decreases in 2-oxoglutarate dehydrogenase activity (KGDH). The open field behavior of Balb/c mice was about 40% less than in CD-1 mice and responded in a qualitatively different manner to thiamin deficiency. The activity of the Balb/c mice increased and then decreased with thiamin deficiency. The activity of 3 month old mice peaked on day 6 (126% of initial score), whereas 10 and 30 month mice showed a much greater increase (about 175% of initial scores), but on day 7. Although the activity of the thiamin dependent enzyme transketolase (TK) was affected similarly at all ages, the activity of KGDH in the aged brain was more sensitive to thiamin deficiency than in the young; KGDH activity declined 41%, 57% or 74% at 3, 10, or 30 months, respectively. Thus, the current mouse model is an attractive one to study the interaction of thiamin deficiency with aging.

Selective alteration of mouse brain neurotransmitter release with age.

The release of acetylcholine (ACh), glutamate (GLU) and dopamine (DA) from various brain regions was investigated in young (3 month) and old (30 month) Balb/c mice. Aging increased the basal release of GLU (77%) and DA (29%) in striatum and GLU in hippocampus (94%); the concentrations of these neurotransmitters in the media after K+ stimulation were unaltered by aging. Although the basal release of ACh was not altered by age, K+-stimulated ACh release was reduced in striatum. The age-related increases in basal GLU and DA release may be important in the pathophysiology of cell death during aging.

Locomotor activity as a predictor of times and dosages for studies of nicotine's neurochemical actions.

Nicotine's action on the central nervous system is complex and likely involves an interaction of neurotransmitters. To determine the time after administration of nicotine and dosage for neurochemical studies, locomotor activity of CD-1 mice was determined at 5 min intervals between 0-60 min. A low nicotine dosage (0.05 mg/kg) did not alter activity 5-15 min after drug injection, but increased activity 28% at 15-25 min post-injection. A high dosage (0.8 mg/kg) reduced total distance 62% and rearing 87% at 5-15 min; at 15-25 minutes total distance declined 56% and rearing 69%; all measures returned to control values after 30 minutes; rearing then increased at 40 min after nicotine. Pretreatment (15 min before nicotine) with mecamylamine (1.0 mg/kg), but not hexamethonium (1.0 mg/kg), prevented the depressant effect of nicotine. Dopamine (DA) and its metabolites as well as acetylcholine (ACh) synthesis were measured at the point of nicotine's maximal depressant action. Striatal levels of dihydroxyphenylacetic acid (DOPAC) were increased and ACh utilization was reduced in striatum (-25%) and cortex (-24%) 10 min after nicotine (0.8 mg/kg). Mecamylamine, while preventing the depressant effect of nicotine on locomotor activity, did not alter its effects on DA metabolism. These results demonstrate that the behavioral outcome of acute nicotine treatment is time and dose-dependent. Nicotine's depressant action appears not to be due to altered DA but may be related to changes in carbohydrate and acetylcholine metabolism.

Effect of decreased oxygen on in vitro release of endogenous 3,4-dihydroxyphenylethylamine from mouse striatum.

The effects of hypoxia on release of endogenous 3,4-dihydroxyphenylethylamine (DA, dopamine) were investigated in mouse striatal slices. Following a 60-min preincubation, potassium increased DA release 12 times between zero and 1 min. By 10 min, uptake processes exceeded release and DA levels in the media decreased. Hypoxia (low oxygen) and anoxia (no oxygen) increased DA in the media by 120 and 205%, respectively, but did not alter dihydroxyphenylacetic acid concentrations. Under similar conditions, anoxia increased [3H]DA uptake eight-fold. For the uptake studies, the amount of DA added to the media was critical; in the presence of high concentrations of DA, anoxia reduced reuptake. Regardless of exogenous DA, hypoxia and anoxia increased extracellular DA, which may play a role in ischemic cell damage.

Behavioral and neurochemical correlates of morphine and hypoxia interactions.

Decreased oxygen availability (hypoxia) impairs the synthesis of dopamine and serotonin in parallel with a decline in open-field behavior. If hypoxic-induced deficits in dopamine and serotonin metabolism are physiologically important, then stimulation of their synthesis may help reverse hypoxic-induced neurochemical and behavioral deficits. Acute morphine sulfate (50 mg/kg) increased dihydroxyphenylacetic acid/dopamine ratios (DOPAC/DA) (+20%), the conversion of [3H]tyrosine to [3H]dopamine (+73%) and open-field activity (+130%) in CD-1 male mice. However, morphine failed to significantly alter the incorporation of [3H]tryptophan to [3H]serotonin. Morphine antagonized the hypoxic-induced impairment of dopamine metabolism and locomotor activity. DOPAC/DA ratios of hypoxic animals that were treated with morphine were identical to controls, and conversion rates of [3H]tyrosine to [3H]dopamine were increased. Total distance in an automated activity monitor following the combination of morphine and hypoxia increased 79% compared to a 48% decrease with hypoxia alone. These results suggest that both hypoxia and morphine alter the dopaminergic system, but in opposite directions. These interactions may help to explain why morphine is able to ameliorate hypoxic-induced changes in behavior.

Monoamine neurotransmitter metabolism and locomotor activity during chemical hypoxia.

The effects of hypoxia on metabolism of 5-hydroxytryptamine (5-HT or serotonin) and 3,4-dihydroxyphenylethylamine (DA or dopamine) were compared with those on open-field activity in male CD-1 mice. Chemical hypoxia was induced with NaNO2. Hypoxia did not alter striatal concentrations of DA, 5HT, Trp, Tyr, 5-hydroxyindoleacetic acid, or homovanillic acid. However, NaNO2 (75 mg/kg) reduced the rates of conversion of [3H]Tyr to [3H]DA (-41%) and [3H]Trp to [3H]5-HT (-39%). Hypoxia also reduced dihydroxyphenylacetic acid (DOPAC) levels (-27%) and DOPAC/DA ratios (-20%). Open-field behavior, as measured in an automated activity monitor, decreased in a dose-dependent fashion with 75-150 mg/kg of NaNO2 (-35 to -90%). Comparison with previous studies suggests that the syntheses of dopamine, serotonin, and the amino acids are equally vulnerable to hypoxic insults but may be less sensitive than the synthesis of acetylcholine.

Automated method to estimate catecholamine and indoleamine content and turnover rates.

A double-label isotopic method for estimation of the rate of formation of serotonin (5-HT) and dopamine (DA) in mouse striatum, hippocampus and cortex was standardized. Mice received an intravenous pulse injection of [3H]tryptophan (TRP) and [3H]tyrosine (TYR) at 2.5, 5, 10 or 20 min before sacrifice by microwave irradiation. Compounds of interest were separated by automated high-performance liquid chromatography and their contents were determined by electrochemical detection. Programmed collection of the TYR, DA, 5-HT and TRP peaks allowed determination of their radioactivity by liquid scintillation. Conversion of [3H]TYR to [3H]DA was nearly ten times greater in striatum than cortex, whereas the formation of [3H]5-HT from [3H]TRP was similar in striatum, cortex and hippocampus.

Monoamines and effects of chlordiazepoxide on the corticosterone response to stress.

Administration of chlordiazepoxide (CDP) (5 mg/kg) decreased plasma corticosterone (CS) levels of mice exposed to noise stress and increased CS levels in nonstressed mice (20 and 40 mg/kg). The increase in CS may be related to an observed decrease in locomotor activity. Quipazine (0.5 mg/kg), a serotonergic agonist, failed to alter CS levels in animals without prior drug treatment and attenuated the rise in CS of nonstressed mice. These results contradicted a possible serotonergic facilitatory effect on CS regulation. Administration of apomorphine (0.5 mg/kg) or clonidine (0.1 mg/kg) prevented the significant reduction of CS levels in stressed mice treated with low doses of CDP. Apomorphine, alone, increased CS of nonstressed mice to levels approximating those of vehicle-treated stressed mice. Thus, a norepinephrine inhibitory effect was not evident and possible dopaminergic involvement in CS response regulation was suggested. It seemed likely that the effect of CDP on the hypothalamic-pituitary-adrenal system was dependent upon the endocrine state of the animal. Mice exposed to stress and exhibiting higher levels of CS were affected differently by CDP treatment than their nonstressed counterparts.