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.

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.

Health risks from increases in methylmercury exposure.

Our present knowledge of the human health effects of methylmercury exposure is derived from study of major outbreaks of human poisonings in Japan and Iraq and experimental studies on primates. Methylmercury readily passes through such physiological barriers as the blood-brain barrier, blood-testes barrier, and the placenta. Its major pathological effects are on the nervous and reproductive systems and the developing embryo/fetus. The neurotoxicity of methylmercury is well established in both humans and non-human primates. Lesions in the cerebral and cerebellar gray matter consist of necrosis and lysis of neurons, phagocytosis and gliosis. The changes are most prominent in the deep sulci and may have a vascular component. A late effect is cerebral atrophy. At high dose levels the liver, kidneys, and other organs may also have degenerative changes. Although not yet described in humans, a major effect of exposure of female primates is an adverse effect on pregnancy. Maternal female M. fascicularis blood mercury levels above 1 ppm are associated with a decreased pregnancy rate and increased abortion rate. To date our experimental data lack sufficient numbers to detect infrequent pregnancy effects below 1 ppm. Preliminary studies also reveal that methylmercury may also decrease the number and function (swim speed) of sperm. Both human and primate studies demonstrate deleterious effects of methylmercury on the developing embryo/fetus. Autopsies on human and primate infants reveal retarded brain development and the occurrence of a cerebral palsy-like behavior in the newborns, whereas the mother may be free of signs and symptoms of methylmercury toxicity. The fetal blood level of mercury is higher than the maternal level.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

Laser light-scattering study of the toxic effects of methylmercury on sperm motility.

An in vitro study was designed using the laser light-scattering technique to obtain further information on the dose-effect relationship of methylmercury on sperm motility. The technique provided a quantitative evaluation of sperm swimming speed. Semen samples were collected from normal male Macaca fascicularis monkeys by anal electroejaculation. Methylmercury was added to aliquots of sperm suspensions in BWW medium in doses of 10, 5, 2, and 1 ppm. After 3 hours, the relative speed was 35%, 59%, 69%, and 92% of the corresponding controls at doses of 10, 5, 2, and 1 ppm, respectively. The percentage of motile spermatozoa decreased significantly at 10 ppm. By microscopic observation abnormal motility was detected at 5 and 10 ppm, especially after 20 to 40 minutes. Head movement increased from side to side, and many spermatozoa developed coiled tails. The technique proved useful for defining the dose-effect relationship of methylmercury and sperm swimming speed.

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.

Methylmercury effects on reproduction and offspring size at birth.

This article describes a study of the toxic, reproductive, and developmental effects of chronic methylmercury (MeHg) exposure in Macaca fascicularis monkeys. Adult and infant monkeys were studied using procedures to assess maternal and newborn blood Hg concentrations, menstrual cyclicity, conception rate, reproductive outcome, maternal toxicity, and offspring size at birth. Maternal intakes of 0, 50, 70, or 90 micrograms/kg/d MeHg hydroxide were studied. Maternal blood Hg concentrations reached equilibrium by 10 weeks of exposure. The half-life of blood Hg for adult females ranged from 15 to 40 days (mean = d) and did not vary with dose. Maternal MeHg exposure did not affect the length of the menstrual cycle or the conception rate. Maternal MeHg exposure did significantly reduce the number of viable deliveries at blood Hg concentrations above 1.5 ppm. Maternal blood Hg concentrations at delivery were significantly lower than newborn concentrations. No effect of maternal MeHg exposure on offspring size at birth was observed. Maternal toxicity was related to blood Hg concentrations above 2.0 ppm following approximately one year of exposure. Results indicate that MeHg exposure can affect reproductive outcome at levels that do not cause overt toxicity.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Methylmercury exposure and reproductive dysfunction in the nonhuman primate.

Macaca fascicularis females were monitored daily through four menstrual cycles then orally administered 0, 50, or 90 micrograms/kg/day methylmercury hydroxide (MeHg). Females were monitored through four additional menstrual cycles and after approximately 124 days of MeHg treatment were time-mated to nontreated males. MeHg treatment did not affect menstrual cycle or menses length. The relationship between dosage of MeHg and reproductive outcome approached but did not reach statistical significance. Reproductive failure (i.e., nonconception, abortion) was, however, related to a significantly higher blood Hg concentration than reproductive success for MeHg-treated females. The offspring of MeHg-treated females tended to be smaller than control infants, but no statistically significant decrease in gestation, birthweight, or crown-rump length was observed. None of the females receiving MeHg exhibited signs of MeHg toxicity during breeding or pregnancy. Daily treatment with 90 micrograms/kg/day MeHg for nearly 1 year, however, produced signs of toxicity in four of seven females. Toxicity was related to increased maternal size, duration of MeHg treatment, and a blood Hg concentration of 2.3 to 2.8 ppm.

Effects of methyl mercury on sperm oxygen consumption.

Oxygen consumption and percent motile spermatozoa were determined for semen samples from healthy male monkeys Macaca fascicularis. Methyl mercury was added to the samples, in the oxygen measurement chamber, at a concentration of 9 p.p.m. for 15 min. and then increased to 15 p.p.m. for 15-30 min. Oxygen consumption and percent motility were determined during each period. Methyl mercury addition resulted in decreased sperm motility, but we did not detect any inhibition in the rate of oxygen consumption accompanying the decreased motility. On the contrary, the rate of oxygen consumption increased at 15 p.p.m. within 15 min., while the sperm motility was close to zero. Antimycin inhibited the increased rate of oxygen consumption demonstrating the mitochondrial source of this increased rate. Oligomycin also inhibited the increased rate of oxygen consumption due to methyl mercury, thus excluding the possibility of uncoupling of mitochondrial oxidative phosphorylation. Interference with mitochondrial energy production does not seem to be the primary mechanism of methyl mercury-induced decreased spermatozoal motility. Methyl mercury interference with the dynein/microtubule sliding assembly now seems to us to be a more tenable hypothesis.