Power spectrum analysis of EEG in a translational nonhuman primate model after chronic exposure to low levels of the common marine neurotoxin, domoic acid.

Domoic acid (DA), the focus of this research, is a marine algal neurotoxin and epileptogen produced by species in the genus Pseudo-nitzschia. DA is found in finfish and shellfish across the globe. The current regulatory limit for DA consumption (20 ppm in shellfish) was set to protect humans from acute toxic effects, but there is a growing body of evidence suggesting that regular consumption of DA contaminated seafood at or below the regulatory limit may lead to subtle neurological effects in adults. The present research uses a translational nonhuman primate model to assess neurophysiological changes after chronic exposure to DA near the regulatory limit. Sedated electroencephalography (EEG) was used in 20 healthy adult female Macaca fascicularis, orally administered 0.075 and 0.15 mg DA/kg/day for at least 10 months. Paired video and EEG recordings were cleaned and a Fast Fourier Transformation was applied to EEG recordings to assess power differences in frequency bands from 1-20 Hz. When DA exposed animals were compared to controls, power was significantly decreased in the delta band (1-4 Hz, p < 0.005) and significantly increased in the alpha band (5-8 Hz, p < 0.005), theta band (9-12 Hz, p < 0.01), and beta band (13-20 Hz, p < 0.05). The power differences were not dose dependent or related to the duration of DA exposure, or subtle clinical symptoms of DA exposure (intentional tremors). Alterations of power in these bands have been associated with a host of clinical symptoms, such as deficits in memory and neurodegenerative diseases, and ultimately provide new insight into the subclinical toxicity of chronic, low-dose DA exposure on the adult primate brain.

Nonhuman primates as animal models for toxicology research.

There is a long history for the use of nonhuman primates in toxicological research. This unit reviews applications in reproductive toxicology and teratology, neural toxicology and neurobehavioral toxicology, immunotoxicology, respiratory (lung) toxicology, and chemical carcinogenesis.

Methods for studying nonhuman primates in neurobehavioral toxicology and teratology.

The behavioral repertoire of nonhuman primates is highly evolved and includes advanced problem-solving capabilities, complex social relationships, and sensory acuity equal or superior to humans. These factors make nonhuman primates valuable animal models for studies of the functional effects of neurotoxicants. This review provides descriptions of tests designed to study learning, memory, schedule-controlled behavior, information processing, social behavior, sensory functioning, and visual-motor coordination and/or visuospatial orientation in macaque monkeys. Whenever possible, the results of studies in primate behavioral toxicology are provided for individual test measures. The primate model is especially useful for studies of developmental exposures because monkeys, like humans, have relatively prolonged periods of gestation, infancy, and adolescence. In recognition of this, a special section is provided for tasks that are specifically designed to study behavioral processes in infant monkeys.

Mechanisms underlying Children's susceptibility to environmental toxicants.

An important public health challenge has been the need to protect children's health. To accomplish this goal, the scientific community needs scientifically based child-specific risk assessment methods. Critical to their development is the need to understand mechanisms underlying children's sensitivity to environmental toxicants. Risk is defined as the probability of adverse outcome and when applied to environmental risk assessment is usually defined as a function of both toxicity and exposure. To adequately evaluate the potential for enhanced health risks during development, both child-specific factors affecting toxicity and exposure need to be considered. In the first section of this article, example mechanisms of susceptibility relevant for toxicity assessment are identified and discussed. In the second section, examples of exposure factors that help define children's susceptibility are presented. Examples of pesticide research from the newly funded Child Health Center at the University of Washington will be given for illustration. The final section discusses the importance of putting these considerations of children's susceptibility into an overall framework for ascertaining relevancy for human risk assessment.

Fixed interval/fixed ratio performance in adult monkeys exposed in utero to methylmercury.

Previous studies in monkeys and rodents have shown the fixed interval/fixed ratio (FI/FR) schedule to be a sensitive indicator of neurotoxicity. In the present study, monkeys (Macaca fascicularis) were exposed in utero to methylmercury (MeHg). Maternal doses of MeHg of 50, 70, or 90 micrograms/kg b.wt./day resulted in infant blood mercury levels at birth ranging from 1.04 to 2.45 ppm. Monkeys were tested on a multiple FI/FR schedule of reinforcement at 8-10 years of age. Four FI/FR cycles were run per session. Pause time and run rate were calculated for FI and FR components, as well as FI quarter-life and local FI response rates. MeHg treatment and sex effects were investigated by fitting a linear orthogonal polynomial regression to each monkey's profile across sessions and performing two-way ANOVAs on the resulting linear and intercept terms. There were no treatment-related effects on either the FI or FR component for pause time or run rate. Analysis of the quarter-life revealed a significant treatment by sex effect as well as a main effect for sex. Post hoc t-tests revealed a significant difference in quarter-life of treated male and female monkeys and a marginal difference between treated and control males. The FI run rate of the male monkeys was significantly greater than that of the female monkeys whereas the FR run rate of the males was marginally greater. These results indicate that there may be a differential effect of MeHg on male and female monkeys, which could be interpreted as an effect on temporal discrimination. Overall, adult monkeys exposed to in utero MeHg exhibited a very limited sex-related effects on the FI/FR intermittent schedule of reinforcement.

Changes in the number of astrocytes and microglia in the thalamus of the monkey Macaca fascicularis following long-term subclinical methylmercury exposure.

The effects of long-term subclinical exposure to methylmercury on the number of neurons, oligodendrocytes, astrocytes, microglia, endothelial cells and pericytes within the thalamus from the left side of the brain of the monkey Macaca fascicularis has been determined by use of the Optical Volume Fractionator stereological method. The accumulated burden of inorganic mercury (IHg) within these same cell types has been determined by autometallographic methods. Four groups of monkeys were exposed to methylmercury (MeHg; 50 micrograms Hg/kg body weight/day) by mouth for 6 months, 12 months, 18 months, or 12 months followed by 6 months without exposure (clearance group). Neurons, oligodendrocytes, endothelia, and pericytes did not show a significant change in cell number for any exposure group. Astrocyte cell number exhibited a significant decline for both the 6 month and clearance exposure groups. The microglia, in contrast, showed a significant increase in the 18 month and clearance exposure groups. Results from mercury speciation and quantification analysis of contralateral matched samples from the thalamus of the right side of the brain from these same monkeys indicated that MeHg concentrations plateaued at around 12 months exposure, whereas the inorganic levels, presumably derived from demethylation of MeHg, continued to increase throughout all exposure durations. Autometallographic determination of the distribution of IHg by cell type indicates that both the astrocytes and microglia contain substantially elevated IHg deposits relative to all other cell types. The data suggest that the inorganic mercury present in the brains, accumulating after long-term subclinical methyl mercury exposure, may be a proximate toxic form of mercury responsible for the changes within the astrocyte and microglial populations.

Demethylation of methyl mercury in different brain sites of Macaca fascicularis monkeys during long-term subclinical methyl mercury exposure.

Total (T-Hg) and inorganic (I-Hg) mercury concentrations were determined in specific brain sites (cerebellum, occipital pole, pons, motor strip, frontal pole, temporal pole, thalamus, and pituitary) of female Macaca fascicularis monkeys exposed to daily peroral doses (50 micrograms Hg/kg body weight) of methyl mercury (MeHg) for 6, 12, or 18 months, or to continuous iv infusion of HgCl2 (200 micrograms Hg/kg body wt). In normal weight monkeys (2.4-4.1 kg body wt), the average concentration of MeHg (calculated as T-Hg minus I-Hg) was about the same in all brain sites, except the pituitary--3.0 micrograms Hg/g at 6 months, 4.2 micrograms/g at 12 months, and 4.3 micrograms Hg/g at 18 months. MeHg concentrations in the pituitary were about 50% of those in the other brain sites. In a group of monkeys that were kept unexposed for 6 months following 12 months of MeHg exposure, T 1/2 for MeHg was about 37 days in all brain sites, with the exception of the pituitary, where it was shorter. The concentration of I-Hg increased in all brain sites, but especially in the thalamus and pituitary, with the time of MeHg exposure. In most brain sites, I-Hg constituted about 9% of T-Hg at 6 and 12 months, and 12% of T-Hg at 18 months. In the pituitary, I-Hg increased from 20% of T-Hg at 6 months to 46% at 18 months. Elimination T 1/2 for I-Hg was extremely long, 230-540 days in most brain sites and considerably longer in the thalamus and pituitary. The concentration of I-Hg in the thalamus did not decrease during the clearance period (6 months), while I-Hg in the pituitary continued to increase in spite of no additional exposure. The MeHg exposed monkeys had several times higher I-Hg concentrations in the brain than monkeys exposed to HgCl2, indicating that I-Hg was formed by demethylation of MeHg in the brain, and not by brain uptake of I-Hg formed by demethylation elsewhere in the body. There were large variations in the relative concentration of I-Hg between individual monkeys, but not between brain sites (except thalamus and pituitary). Obese monkeys (5.0-6.1 kg body wt) exposed to MeHg had higher concentrations of both MeHg and I-Hg than normal weight monkeys in all brain sites, except in the pituitary.

Autometallographic determination of inorganic mercury distribution in the cortex of the calcarine sulcus of the monkey Macaca fascicularis following long-term subclinical exposure to methylmercury and mercuric chloride.

The distribution of accumulated inorganic mercury deposits in the cortex of the calcarine sulcus of adult female Macaca fascicularis following long-term subclinical exposure to methyl-mercury (MeHg) and mercuric chloride (inorganic mercury-IHg) has been determined by autometallography. Four groups of monkeys were exposed to MeHg (50 micrograms Hg/kg body wt/day) by mouth for 6, 12, and 18 months or 12 months followed by 6 months without exposure (clearance group). A fifth group of monkeys was administered inorganic mercury (as HgCl2; 200 micrograms Hg/kg body wt/day) for 3 months by constant rate intravenous infusion via an indwelling catheter. Staining of IHg deposits in the MeHg-exposed groups increased for all cell types with increased length of exposure. The astrocytes and microglia in the MeHg exposure groups contained the largest deposits of IHg. Neurons in the 6-month MeHg exposure group were either not labeled or contained very fine deposits of IHg. The frequency of labeled neurons increased somewhat in the 12-month and clearance exposure groups. Virtually all neurons in the 18-month exposure group contained labeled deposits of IHg; however, these total deposits were considerably smaller than those present within the astrocytes and microglia. The majority of endothelial cells and pericytes did not contain notable mercury deposits, although scattered individual cells were heavily labeled. Labeled oligodendrocytes were relatively rare in all MeHg-exposed groups. Gitter cells, primarily located in a perivascular position, were common in the 12-month, 18-month, and clearance groups and many of these were found to be heavily labeled. The staining of mercury deposits in the IHg-exposed animals was low compared to the MeHg-exposed groups. The astrocytes and microglia were the primary cell types labeled. It is concluded that the astrocytes, and possibly microglia, are the primary location of the demethylation of MeHg into IHg within the cortex of the calcarine sulcus.

Health risks associated with prenatal metal exposure.

A symposium entitled Health Risks Associated with Prenatal Metal Exposure was held at the 33rd Annual Meeting of the Society of Toxicology (SOT) in Dallas, Texas. The symposium was cosponsored by the Metals and Reproductive and Developmental Specialty Sections of SOT and was designed to elaborate the health risks associated with in utero exposure to metals commonly found in the workplace and/or ambient environment on the mother and developing offspring. Epidemiological and toxicological evidence that demonstrates the health effects and underlying mechanisms associated with exposure to arsenic (As), lead (Pb), and methyl mercury (MeHg) were discussed, as well as the legal ramifications and personal implications associated with prenatal metal exposure. The following is a summary of each of the individual presentations.

Increases in the number of reactive glia in the visual cortex of Macaca fascicularis following subclinical long-term methyl mercury exposure.

The number of neurons, astrocytes, reactive glia, oligodendrocytes, endothelia, and pericytes in the cortex of the calcarine sulcus of adult female Macaca fascicularis following long-term subclinical exposure to methyl mercury (MeHg) and mercuric chloride (inorganic mercury; IHg) has been estimated by use of the optical volume fractionator stereology technique. Four groups of monkeys were exposed to MeHg (50 micrograms Hg/kg body wt/day) by mouth for 6, 12, 18, and 12 months followed by 6 months without exposure (clearance group). A fifth group of monkeys was administered IHg (as HgCl2; 200 micrograms Hg/kg body wt/day) by constant rate intravenous infusion via an indwelling catheter for 3 months. Reactive glia showed a significant increase in number for every treatment group, increasing 72% in the 6-month, 152% in the 12-month, and 120% in the 18-month MeHg exposed groups, and the number of reactive glia in the clearance group remained elevated (89%). The IHg exposed group showed a 165% increase in the number of reactive glia. The IHg exposed group and the clearance group had low levels of MeHg present within the tissue; however, the level of IHg was elevated in both groups. These results suggest that the IHg may be responsible for the increase in reactive glia. All other cell types, including the neurons, showed no significant change in number at the prescribed exposure level and durations. The identities of the reactive glial cells and the implications for the long-term function and survivability of the neurons due to changes in the glial population following subclinical long-term exposure to mercury are discussed.

Neurotoxic effects of gasoline and gasoline constituents.

This overview was developed as part of a symposium on noncancer end points of gasoline and key gasoline components. The specific components included are methyl tertiary butyl ether, ethyl tertiary butyl ether, tertiary amyl methyl ether, butadiene, benzene, xylene, toluene, methyl alcohol, and ethyl alcohol. The overview focuses on neurotoxic effects related to chronic low-level exposures. A few general conclusions and recommendations can be made based on the results of the studies to date. a) All the compounds reviewed are neuroactive and, as such, should be examined for their neurotoxicity. b) For most of the compounds, there is a substantial margin of safety between the current permissible exposure levels and levels that would be expected to cause overt signs of neurotoxicity in humans. This is not the case for xylene, toluene, and methanol, however, where neurologic effects are observed at or below the current Threshold Limit Value. c) For most of the compounds, the relationship between chronic low-level exposure and subtle neurotoxic effects has not been studied. Studies therefore should focus on examining the dose-response relationship between chronic low-level exposure and subtle changes in central nervous system function.

Effects of in utero methylmercury exposure on a spatial delayed alternation task in monkeys.

Adult female monkeys (Macaca fascicularis) were exposed to 0, 50, 70, or 90 micrograms/kg/day of methylmercury prior to and throughout pregnancy and produced 11, 9, 2, and 2 infants, respectively. At birth, blood mercury levels of treated infants ranged from 1.04 to 2.46 ppm. At approximately 7 to 9 years of age, the monkeys were trained by successive approximation to respond on a lit button for a small amount of apple juice. The monkeys were then trained on a 0.1-sec spatial delayed alternation task to a specified criterion of performance. This was followed by 10 sessions each of fixed delay times of 0.5, 1, 3, 5, and 10 sec, followed by 20 sessions containing variable delay times of 0.1 to 15 sec. Data from all treated monkeys were combined. There were no differences between treated and control monkeys in initial button training or number of sessions to reach criterion on 0.1-sec delay procedure. On the fixed delay sessions, the treated monkeys had significantly more correct trials, and fewer incorrect responses, perseverative responses, and delay responses than controls. There were no differences between the treated and control monkeys on performance on the variable delay schedule. Results from this study indicate that in utero methylmercury exposure did not adversely affect the spatial memory of adult monkeys when tested on a delayed alternation task and may have facilitated performance on this task.

Pathways of human exposure to arsenic in a community surrounding a copper smelter.

Several studies have found elevated levels of urinary arsenic among residents living near a copper smelter in Tacoma, Washington. To assess pathways of exposure to arsenic from the smelter, biological and environmental samples were collected longitudinally from 121 households up to 8 miles from the smelter. The concentration of inorganic and methylated arsenic compounds in spot urine samples was used as the primary measure of exposure to environmental arsenic. Urinary concentration of arsenic dropped off to a constant background level within one-half mile of the smelter in contrast to environmental concentrations, which decreased more steadily with increasing distance. Among all age-sex-specific groups in all areas, only children ages 0-6 living within one-half mile of the smelter had elevated levels of arsenic in urine. A separate analysis of data for these children suggests that hand-to-mouth activity was the primary source of exposure. Inhalation of ambient air and resuspension of contaminated soil were not important sources of exposure for children or adults.

Methylmercury developmental neurotoxicity: a comparison of effects in humans and animals.

A qualitative and quantitative comparison of the neuropathological and neurobehavioral effects of early methylmercury (MeHg) exposure is presented. The focus of the qualitative comparison is the examination of how specific end-points (and categories of behavioral functions) compare across species. The focus of the quantitative comparison is the investigation of the relationship between MeHg exposure, target-organ dose and effects in humans and animals. The results of the comparisons are discussed in the context of the adequacy of the proposed EPA neurotoxicity battery to characterize the risk of MeHg to humans. The comparisons reveal several qualitative and quantitative similarities in the neuropathological effects of MeHg on humans and animals at high levels of exposure. Reports of neuropathological effects at lower levels are available for animals only, precluding any comparison. At high levels of exposure, specific neurobehavioral end-points affected across species are also similar. Effects at lower levels of exposure are similar if categories of neurobehavioral functioning are compared. Changes in the EPA test battery consistent with the results of the comparisons are discussed.

Methylmercury effects on the social behavior of Macaca fascicularis infants.

Observations of the social behavior of Macaca fascicularis exposed in utero to methylmercury (MeHg) and nonexposed control infants were performed as part of a study of the toxic, reproductive and developmental effects of maternal MeHg intake. Infants were tested twice weekly from 2 weeks to 8 months of age. Data were summarized into 6 categories of social behavior and 7 categories of nonsocial behavior. Analysis of the most prevalent behavior indicated that MeHg-exposed offspring exhibited a decrease in social play behavior and a concomitant increase in nonsocial passive behavior. The MeHg effect on social play behavior tended to decrease with age, while the group differences in nonsocial passive behavior tended to increase. The results indicate that maternal intake of MeHg during pregnancy can affect the social development of infant primates by suppressing social interactions and increasing nonsocial behavior.

Kinetics of methyl mercury in blood and brain during chronic exposure in the monkey Macaca fascicularis.

The disposition parameters derived from a compartmental model kinetic analysis of blood Hg levels in nonpregnant, adult female Macaca fascicularis given daily doses of MeHg did not vary with either dosage level (50, 70 or 90 micrograms MeHg/kg b.wt.day) or duration of exposure (up to 507 day). In contrast, blood clearance of Hg in pregnant females was dose-dependent; it being higher at the 90 micrograms MeHg/kg b.wt.day than at the lower dosage levels. Hg levels in the brain of adult fascicularis relative to blood Hg also increased at the highest level of exposure. Blood Hg half-life in neonate fascicularis was similar to half-life in their mothers (adults). Finally, the regional distribution of mercury in the brains of adult and neonate fascicularis exposed to low and intermediate levels of MeHg resembles the reported distribution of mercury in the brains of adult and neonate humans environmentally exposed to MeHg. Consequently, M. fascicularis may be an especially appropriate animal model for studying the neurotoxic mechanisms of chronic methyl mercury exposure.

Diagnosis of early pregnancy by ultrasound in Macaca fascicularis.

Real-time ultrasonography was used to detect early pregnancy in 32 longtailed macaques (Macaca fascicularis). In 92% of the successful conceptions, a correct diagnosis was made. The earliest sign of pregnancy was an intrauterine ringlike structure (11 days). A "line swelling" (14 days) preceded definite fetal echoes (21 days), and fetal heart motion (30 days) proved fetal viability. Ultrasound is a rapid, noninvasive, and relatively cost-effective method of diagnosing and monitoring early pregnancy in M. fascicularis.

Visual recognition memory deficits in methylmercury-exposed Macaca fascicularis infants.

Infant Macaca fascicularis exposed prenatally to maternal subclinical levels of methylmercury (MeHg) and their nonexposed controls were administered a test of visual recognition memory beginning at 210 days postconception (mean postnatal age = 51.88 days, SD = 5.30). The test consisted of a series of problems in which two identical 35 mm slides of a monkey's face were presented for a study period, followed by a test trial in which the previously exposed stimulus was paired with a novel one, and the looking time to each was recorded. The nonexposed group showed differential visual attention to the novel stimuli, indicating visual recognition abilities. The exposed group's visual attention to the novel stimuli was random. These results, in conjunction with earlier findings, suggest that prenatal MeHg exposure is associated with impaired visual recognition memory performance.

Biologic variables in the hair uptake of methylmercury from blood in the macaque monkey.

The total mercury (Hg) in hair and blood of 45 young healthy adult female Macaque fascicularis given 0, 50, 70, or 90 micrograms MeHg/kg body wt orally in apple juice daily revealed a close and constant ratio between blood Hg and hair. The amount of hair Hg does not increase with time (maximum period of observation 490 days) at a given dose level. Also the ratio was unchanged between background and subtoxic dose levels. Individuals at a given dose level with a higher-than-average blood level had a proportionately higher hair level. The Macaque blood/hair ratio is markedly lower than that reported for humans. Pregnancy did not have an appreciable effect on the hair mercury level. Review of the known variables in human and Macaque hair growth and structure does not provide an explanation for the difference. We suggest that an as yet unidentified biological variable(s), possibly circumfollicular blood flow, could account for the difference. This ratio difference notwithstanding, controlled studies on Macaque hair such as this add support for the validity of terminal hair as a trace metal exposure indicator.

Effects of methyl mercury on testicular functions in Macaca fascicularis monkeys.

These studies were performed to investigate the effects of MeHg on testicular function in Macaca fascicularis monkeys. In an in vivo study involving oral treatment of adult males Macaca fascicularis monkeys with MeHg for 20 weeks, changes in spermatozoal production, motility and morphology and in serum testosterone were followed before, during and after treatment. MeHg treatment significantly decreased % motile spermatozoa and scores for sperm speed and forward progression and increased % abnormal sperm tail forms, at sub-neurotoxic levels. The MeHg-induced increase in semen abnormalities was not accompanied by any significant changes in serum levels of testosterone. No consistent histological abnormalities were detected in testicular biopsies from the treated animals at the end of the treatment period. A good recovery pattern was observed for the MeHg effects on sperm motility while this was unclear for the effects on sperm morphology.