Effects of lead on neurophysiological and performance measures: animal and human data.

This paper reports lead-induced changes in neuropsychological measures and behavioral performance measures in monkeys and children. Monkeys were pre- and postnatally exposed to lead via the diet. Blood lead levels at the time of testing were 9.3, 40.3, and 55.7 micrograms/dL in controls, and animals exposed to 350 ppm or 600 ppm lead acetate, respectively. Flash-evoked and brainstem auditory-evoked potentials were recorded in adult animals. Results indicate latency increases in both measures as well as amplitude decreases in the flash-evoked response. Delayed reaction time and serial choice reaction were determined as measures of behavioral performance in lead-exposed school-age children from two lead smelter areas. In addition, pattern-reversal-evoked potentials and nerve conduction velocity were investigated. Neither nerve conduction velocity nor latency of the pattern-reversal-evoked potential were consistently influenced by lead. Of the behavioral measures, serial choice reaction performance revealed a consistent lead-related deficit, which became more pronounced with increasing task difficulty.

Reduced levels of 1,25-dihydroxyvitamin D(3) in rat dams and offspring after exposure to a reconstituted PCB mixture.

Previous studies revealed effects of polychlorinated biphenyls (PCBs) and other polyhalogenated hydrocarbons on steroid hormone levels and hormone-dependent functions including behavior. In the present study serum concentrations of the vitamin D(3) metabolites 25-hydroxycholecalciferol (25-D) and 1,25-dihydroxycholecalciferol (1,25-D) were determined in rat dams and offspring after exposure to a PCB mixture that was reconstituted according to the congener pattern found in human breast milk. Unmated females were exposed to diets adulterated with 0; 5; 20; or 40 mg PCBs/kg diet. Exposure started 50 days prior to mating and was terminated at birth. Gestational exposure reduced serum concentrations of 1,25-D in dams in a dose-dependent manner. Concentration of 25-D was also decreased at the time of delivery, but not at weaning. Determination of 1,25-D in offspring at weaning revealed reductions in both high-exposure groups. Levels of 25-D were diminished only at the highest exposure level. Internal PCB concentrations in adipose tissue and brains exhibited a linear relation to dosages in diet. Concentrations of PCBs in brains were similar in dams and offspring at birth, but decreased at the end of lactation in dams. In offspring, values increased during this period because of continued exposure via the milk. In the adipose tissue, PCB levels were much lower in offspring than in dams. To our knowledge, this is the first report of PCB-induced effects on vitamin D(3) metabolites. In dams, reductions were seen even at the lowest exposure level used. Further studies are needed to evaluate the biological significance of these reductions in pregnant dams and possible consequences for the developing offspring.

Developmental exposure of rats to a reconstituted PCB mixture or aroclor 1254: effects on long-term potentiation and [3H]MK-801 binding in occipital cortex and hippocampus.

The central nervous system is one of the target organs for polychlorinated biphenyls (PCBs). We measured the effects of maternal exposure of Long-Evans rats to a mixture of PCB congeners reconstituted according to the pattern found in human breast milk (reconstituted mixture, RM) on long-term potentiation (LTP) in two brain regions. Exposure of the dams via food started 50 days prior to mating and was terminated at birth. In the first experiment, adult male and female offspring were exposed maternally to 40 mg/kg of the RM or the commercial mixture Aroclor 1254 (A1254). LTP and paired-pulse inhibition were measured in slices of the visual cortex. In addition, the binding of [3H]MK-801 to the N-methyl-D-aspartate (NMDA) receptor-ion channel as well as the [3H]muscimol binding to the GABA-A receptor in membrane preparations from the occipital cortex and hippocampus were determined. LTP as well as [3H]MK-801 binding were significantly reduced in the cortex following PCB exposure, while [3H]MK-801 binding in the hippocampus was not affected. In a succeeding experiment, LTP was determined in cortical and hippocampal slices from rats at postnatal days 10 to 20, following exposure to 0, 5, or 40 mg/kg of the RM. Cortical LTP was significantly affected by the RM while no effects were seen in hippocampal LTP. Taking the two experiments together, PCB exposure significantly reduced LTP, as well as [3H]MK-801 binding, in the cortex and had no effect in the hippocampus. The LTP deficits can only partly be related to the reduction of binding sites to the NMDA receptor; other PCB-induced neurochemical changes have to be assumed.

Inhibition of long-term potentiation in developing rat visual cortex but not hippocampus by in utero exposure to polychlorinated biphenyls.

The neurotoxic potential of polychlorinated biphenyls (PCBs) depends on the structure of the congener as well as on the age of the exposure. We exposed rats prenatally to a coplanar congener (PCB-77) or to a non-coplanar congener (PCB-47) and measured the amount of long-term potentiation (LTP) at postnatal days 11-19 in the visual cortex and hippocampus. While PCB-77 exposure affected LTP statistically significantly in cortical but not hippocampal slices, the exposure to PCB-47 was much less effective.

Maternal exposure to polychlorinated biphenyls inhibits long-term potentiation in the visual cortex of adult rats.

Rats were exposed prenatally to the coplanar congener 3,3',4,4'-tetrachlorobiphenyl (TCB). The amount of long-term potentiation (LTP) was measured in slices from the visual cortex and hippocampus of the adult males as well as of controls. While in the cortical slices from the controls a stable LTP could be induced, LTP was inhibited in the TCB-exposed rats. In hippocampal slices, the amount of LTP was not significantly different between the two groups. Our results suggest that the visual cortex of adult animals is functionally altered by TCB if the exposure takes place during embryonic development.

The effects of lead in laboratory animals and environmentally-exposed children.

Consistent association has been found in 4 independent studies between disruption of visual-motor integration and reaction performance and markers of lead exposure (blood, teeth) in children, but not for intelligence deficit; blood-lead levels were typically below 30 micrograms/dl in these children. In order to demonstrate the persistent nature of Pb-induced neurobehavioral deficit experimentally rats were studied after cessation of dietary Pb-exposure in a variety of behavioral tasks. Persistent retention-deficit was found for visual discrimination-learning and for radical arm-maze performance.

Immunohistochemical localization of neuronal and glial calcium-binding proteins in hippocampus of chronically low level lead exposed rhesus monkeys.

The purpose of this study was to investigate the distribution of the neuronal calcium-binding proteins parvalbumin, calbindin D28k, calretinin and the glial protein S100 in the hippocampus of lead exposed rhesus monkeys. It has been suggested that lead may exert its toxic effects by perturbing the intracellular calcium homeostasis. Lead is able to increase the intracellular Ca2+ concentration and can serve as a calcium substitute. It has been shown that some calcium-binding proteins are capable of binding lead. We tried to find a putative dose-depending relation between long-term low level lead exposure and the expression of the proteins investigated. Rhesus monkeys were pre- and postnatally exposed to 600 mg-350 mg-0 mg lead-acetate in diet for nine years, as described by Lilienthal et al. (1986). After a lead-free period of 32 months animals were sacrificed. Hippocampal paraffin sections were stained for parvalbumin (PV), calbindin D28k (CB), calretinin (CR), and S100 with immunohistochemical methods. The distribution of the neuronal calcium-binding proteins was almost identical for the different exposure groups. The most striking observation was a marked decrease of S100 immunoreactivity in astrocytes in the high lead group. Considering a protective role against high Ca2+ concentration and Pb2+ accumulation respectively the unchanged expression of PV, CB, and CR remains to be clarified. The apparent difference in S100 expression supports the hypothesis that glial cells are the main target of lead toxicity. The reduced expression may indicate a developmental retardation of astroglia.

Persistent decrease of the dopamine-synthesizing enzyme tyrosine hydroxylase in the rhesus monkey retina after chronic lead exposure.

One of the toxic effects of lead in the CNS is an altered functional state of the catecholamine system, especially a reduction in the activity of tyrosine hydroxylase (TH), the rate-limiting enzyme of catecholamine synthesis. Here we report on a lead-induced decrease in TH-content in neurones of the rhesus monkey retina. Rhesus monkeys were pre- and postnatally exposed to 0, 350, or 600 ppm of lead acetate (Pb) in the diet over 9 years. Lead exposure was followed by a 35-month period of lead-free diet. During this period, blood lead levels of the treated animals declined to nearly those of the untreated controls. Subsequently the animals were sacrificed and the retinas processed for TH immunocytochemistry. The fluorescent dye FITC was used to visualise the antibody reaction. Photometric measurements of the fluorescence intensity of stained neurones were made with a laser scanning microscope. In the rhesus monkey retina two types of TH-immunoreactive neurones are present. In the bright fluorescent type, lead exposure resulted in decreased fluorescence intensity and altered the intensity profile of the TH-immunoreactive cells in a dose-dependent manner. In these cells, fluorescence intensity was 0.53 and 0.22 for 350 ppm Pb and 600 ppm Pb respectively when the fluorescence intensity of the untreated controls (0 ppm Pb) is taken as 1. Both lead doses also reduced the number of ascending fibres in the inner nuclear layer and the dense staining of fibres in sublayer 1 of the inner plexiform layer. The weakly fluorescent cell type disappeared to a large extent under 350 ppm Pb treatment and was not detectable in the 600 ppm Pb group. The results demonstrate that lead exposure affects the dopaminergic retinal amacrine cells by reducing the TH-content in these neurones and that this neurotoxic effect persists beyond the end of exposure.

Myopathy: a possible effect of chronic low level lead exposure.

Morphological changes in the central nervous system and other organs have been reported in numerous studies investigating low level lead exposure. To date, however, there are no investigations on the effect of low level lead exposure on striated muscles, although varying neuromuscular changes in different species have been known for years. Rhesus monkeys were exposed pre- and postnatally to lead acetate in the diet (350 ppm or 600 ppm) over 9 years, followed by a lead free period of 32 months, while a control-group received regular diet. No signs of muscular dysfunction were evident. To elucidate neuromuscular pathomorphology frozen sections of the vastus medialis muscle were processed for routine and enzymohistological staining (Hematoxilin and Eosin, Sudan Black, Gomori, NADH, ATPase). Resin histology was processed for electron microscopy. Morphometric analysis was made with commercial software. Light microscopy revealed dose-related signs of myopathy in the lead-exposed groups. The scatter of fibre diameters was increased, and split fibers and internal nuclei were more frequent. Fibres became separated from each other by copious endomysial connective tissue. Ultrastructural examination showed hydropic mitochondria and a massively dilated sarcotubular system in the 600 ppm group. Dose-related extracellular collagen deposition increased. A heavy fibrosis was seen in the 600 ppm group. These findings are interpreted as myopathical reaction due to chronic low level lead exposure, as there were no signs of neurogenical lesion. It remains unknown how the fibrosis developed. A primary fibrosis could be based upon a developmental delay of satellite cells (expressing metalloproteases for collagen-catabolism). Lead is known to inhibit regular development in many ways if exposure has started prenatally. As the skeletal muscle is a common target of toxicity, the myotoxic effects of chronic low level lead exposure comes into question.

Developmental delay of astrocytes in hippocampus of rhesus monkeys reflects the effect of pre- and postnatal chronic low level lead exposure.

Rhesus monkeys were pre- and postnatally exposed to lead-acetate at 0, 350, or 600 ppm in diet for nine years, followed by a period of lead-free diet for 32 months. During this time blood lead levels declined to normal, but still showed dose-related differences. In behavioral and neurophysiological studies the rhesus monkeys exhibited dose-related cognitive and functional deficits. After sacrifice hippocampal sections were processed for immunohistological staining. GFAP, introduced as a marker of neurotoxicity and Vimentin, which is expressed by immature or reactive astrocytes were investigated. A dose-dependent increase of GFAP due to prenatal and chronic low level lead exposure was not observed. We found a dose-related increase of GFAP-positive radial glia and star-shaped Vimentin-positive astrocytes in the high lead group. We consider these findings as indication of immature astrocytes, which are not able to react with gliosis in respond to pre- and postnatal low level lead exposure. The lack of pronounced glial response due to low level lead exposure may result in a delay of astrocytic differentiation, shown by persistence of radial glia.

Extrapolation from animals to humans: scientific and regulatory aspects.

The necessity to protect humans from the adverse effects of chemicals on structure and function of the developing and/or mature nervous system is increasingly recognized among regulatory bodies throughout the world. Whereas structural changes dominated much of neurotoxicological research in the past, functional markers of neurotoxicity are gaining acceptance as early signs of insult. Among the more ambitious protective efforts are legal requirements of premarket testing for "behaviour-disrupting properties" of chemicals. Assumed or proven validity of cross species extrapolation underlies the use of animals in primary or secondary screening schemes. Two steps must be distinguished here. The first step is endpoint-base or qualitative, whereas the second one is dose-based or quantitative. Species comparisons in terms of endpoints is typically done within a framework of broad functional categories, such as sensory, motivational, cognitive, motor or social functions. Dose-based extrapolation requires knowledge about species differences in terms of toxicokinetics or metabolism in order to arrive at valid translations of dose-response contingencies. Principles of cross-species extrapolation in neurotoxicology will be exemplified by means of representative neurobehavioural and neurophysiological findings for neurotoxic chemicals of environmental concern, for which an adequate data base is available for comparative purposes, such as inorganic lead and polychlorinated biphenyls (PCB).

Impairment of long-term potentiation and learning following chronic lead exposure.

Chronic lead exposure during brain development is known to affect functions of the central nervous system. We exposed rats chronically to low levels of lead at different developmental stages in order to determine the most sensitive periods of exposure. Active avoidance learning and hippocampal long-term potentiation were tested in the same animals. If the exposure period comprised the prenatal and the early postnatal phase and was continued into adulthood, learning as well as long-term potentiation were impaired. Starting the exposure not before 16 days postnatally, however, neither affected learning nor hippocampal potentiation. These results reflect the higher vulnerability of the immature as compared to the mature hippocampus to lead-induced functional deficits.

Lead effects on the brain stem auditory evoked potential in monkeys during and after the treatment phase.

Rhesus monkeys were pre- and postnatally exposed to either 0, 350, or 600 mg lead acetate/kg diet continuously until the age of about 9.75 years. At the age of 8-8.25 years (Experiment 1) and 9.25-9.5 years (Experiment 2) brain stem auditory evoked potentials (BAEPs) were recorded. Blood lead levels at the time of testing were about 5, 35, or 55 micrograms/dl for controls, the 350-mg group and the 600-mg group, respectively. There were no clinical signs of intoxications. Clicks varying in sound pressure level (SPL) and rate were used to elicit BAEPs. In addition, the influence of different levels of masking noise was explored in Experiment 1. Four early prominent waves were detected in accordance with other studies of the monkey BAEP. The most reliable wave was No. II. Latencies in the BAEP exhibited the known dependencies on parametric variation for SPL, stimulus rate, and masking level. The 600-mg group exhibited the longest latencies at all stimulus conditions. Analysis of wave II and IV latencies revealed a significant main effect for lead on wave II. At the rate condition there were also signs of latency decreases in the 350-mg group that did not reach significance. Therefore, repetition rate was varied on all SPLs in Experiment 2 to assess the reliability of this effect because similar observations were reported in lead-exposed children. There was no indication of reduced latencies using this extended design. In contrast, significant lead-induced increases in latencies of waves I, II, and IV were revealed by multivariate ANOVA. The purpose of Experiment 3 was to examine whether these results were dependent on current exposure or persisted after cessation of lead treatment. It started 18 months after the end of lead feeding, when blood lead levels had declined to nearly normal values. The same lead-related effects were detected as in the previous experiments. Taken together, these results indicate consistent prolongations of latencies in the BAEP due to subtoxic lead exposure that are not dependent on current treatment. The results are compared to the effects found in epidemiological studies in lead-exposed children.

Significance of hippocampal dysfunction in low level lead exposure of rats.

Previous reports have suggested a relationship between the neurotoxicity of lead and hippocampal dysfunction. Therefore, a comparison between the behavioral changes induced by lead exposure and by selective destruction of hippocampal neurons should help to clarify whether the intrinsic neurons of the hippocampus are directly influenced by lead. Rats maternally and permanently exposed to lead (750 ppm in the diet as lead acetate) were tested in a radial arm maze and compared with controls and rats with ibotenic acid-induced neuronal depletion in the dorsal hippocampus. Lead-exposed groups showed an impairment in the acquisition performance of the spatial task while hippocampally damaged animals did not. When they were retested 4 weeks after the end of the original acquisition, both groups of lead-exposed and ibotenic acid-treated rats showed a significant deficit in retention. These results suggest that this deficit produced by lead can be due to the damage of the hippocampal neurons but not the impairment observed in the acquisition. We propose that the neurotoxicity of lead is not entirely due to the dysfunction of the dorsal hippocampus and that other areas of the brain should be considered. Both maternally and permanently lead-exposed rats showed a similar degree of deficit in acquisition and retention, suggesting a long-lasting effect of early lead exposure.

Alteration of the visual evoked potential and the electroretinogram in lead-treated monkeys.

Rhesus monkeys were pre- and postnatally exposed to either 0, 350, or 600 ppm lead acetate in lab chow. At the age of 7-7 1/4 years visual evoked potentials (VEP) and electroretinograms (ERG) were recorded. Flashes were used as stimuli. The VEP was taken under two background illuminance conditions. Lead-related decreases in amplitudes and increases in latencies were observed. Effects on amplitudes were more pronounced under the dark condition while latencies were more affected at the bright background level. The ERG was studied during the course of dark adaptation. The increase in amplitudes of the b-wave during the adaptation period was more prominent in lead-exposed subjects than in controls. Oscillatory potentials were not altered by lead. The findings are discussed in terms of the physiological mechanisms underlying these different potentials.

Neuronal depletion of the amygdala resembles the learning deficits induced by low level lead exposure in rats.

The behavioral deficits observed after lead exposure have been related to limbic system dysfunction. In a previous study it was shown that the neurotoxicity of lead could not be explained by the damage of the hippocampus alone. The purpose of the present investigation was to use behavioral comparisons to test the hypothesis that the intrinsic neurons of several nuclei of the amygdala, where lead has been found to accumulate, can be a target of the effects of the metal as well. A group of rats were maternally and permanently exposed to lead (750 ppm in the diet as lead acetate). Another group of equally aged and housed rats, never experimentally exposed to lead, were injected ibotenic acid into the amygdala. All groups plus sham-operated and unoperated controls were tested in the open field, the radial arm maze, and a passive avoidance task. The results showed that lead exposure (both permanent and maternal) and amygdalectomy produced a) no effect on locomotor activity, b) impairments in the acquisition phase of the radial maze, and c) impairments in passive avoidance. These results suggest an involvement of the amygdala in the neurotoxic action of lead, but not as the only brain structure. The deficits in permanently lead-exposed rats are more pronounced than in only maternally-exposed animals suggesting a longlasting, but not totally irreversible effect of early lead exposure.

Sex-dependent effects of maternal PCB exposure on the electroretinogram in adult rats.

The purpose of the present experiment was to evaluate the effects of developmental exposure to polychlorinated biphenyls (PCBs) on the visual system. Pregnant Long-Evans rats were treated with the ortho-chlorinated 2,2',4,4'-tetrachlorobiphenyl and/or with the coplanar 3,3',4,4'-tetrachlorobiphenyl. Total dose of PCBs was 18 mg/kg in all groups. Measurements of the flash-evoked electroretinogram (ERG) started in the offspring at an age of about 200 days. The scotopic b-wave, the maximum potential, and oscillatory potentials were recorded after dark adaptation. Amplitudes of these potentials were reduced in female rats exposed to the coplanar PCB. No differences from controls were found in females of other groups or male rats. The results indicate long-lasting effects on the scotopic ERG after maternal PCB exposure that are sex dependent and congener specific. To our knowledge, this is the first experimental report of PCB-related influences on visual processes.

Behavioral effects following single and combined maternal exposure to PCB 77 (3,4,3',4'-tetrachlorobiphenyl) and PCB 47 (2,4,2',4'-tetrachlorobiphenyl) in rats.

The present study has compared the neurobehavioral effects of two structurally different PCB congeners or their combination in rats. Time-mated Long-Evans rats received daily injections of the coplanar PCB 77 (3,4 3',4'-TCB: 0.5 or 1.5 mg/kg), the di-ortho-chlorinated PCB 47 (2,4,2',4'-TCB: 1.5 mg/kg) or a congener mixture (0.5 mg/kg PCB 77 + 1.0 mg/kg PCB 47) from day 7 to 18 of gestation. The PCB exposure levels in brain and perirenal fat of dams and offspring were determined by GC/ECD on gestational day 19 (GD 19), postnatal day 21 (PND 21), and PND 45. PCB 77 was accumulated to a smaller degree than PCB 47. On GD 19, PCB 77 was found to a greater extent in the brains of the offspring than in the brains of the dams, whereas the level of PCB 47 was almost the same in dams and offspring. The testing of open-field behavior in male rats on PND 18 and PND 70 revealed an altered distribution of activity with enhanced activity in the inner zone in PCB 77-treated rats compared to all other groups, while the overall activity was not changed. Distance traveled and rearing behavior on PND 340 were elevated relative to controls in all PCB-treated groups, indicating age-related effects of maternal exposure. A step-down passive avoidance task revealed decreased latencies in the PCB 77 and combined exposure groups on PND 80. Only PCB 77-treated animals showed increased latencies on PND 100 on the haloperidol-induced catalepsy test. These results indicate long-term effects of maternal exposure to PCB 77 on emotional and motor functions. At the dose levels used in the present experiments, the two congeners given in combination did not cause additive or synergistic effects. Instead, concurrent exposure to PCB 47 seemed to counteract PCB 77-induced changes in the pattern of activity.

Behavioral effects of maternal exposure to an ortho-chlorinated or a coplanar PCB congener in rats.

Polychlorinated biphenyls (PCBs) are still of environmental concern. Neurotoxic effects were described after developmental exposure to PCB mixtures and single congeners. The purpose of the present experiment was to compare the behavioral effects of the coplanar congener 3,4,3',4'-tetrachlorobiphenyl with the ortho-chlorinated 2,4,2',4'-tetrachlorobiphenyl. Female Wistar rats were exposed with a subtoxic dose of 1 mg/kg b.w. of 3,4,3',4'-tetrachlorobiphenyl, 2,4,2',4'-tetrachlorobiphenyl or the vehicle during gestation from day 7 to 18. There were significant lower concentrations of 3,4,3',4'-TCB than of 2,4,2',4'-TCB in dams and offspring at gestational day 19. Decreases from gestational day 19 (F 19) to postnatal day 21 (PND 21) were only observed in the adipose from dams exposed to 2,4,2',4'-TCB. The following behavioral tests were conducted in the offspring: locomotor activity in the open field, spatial learning in the radial arm maze, catalepsy induced by the dopamine receptor blocker haloperidol, and passive avoidance learning at PND 25, PND 95, PND 180, and PND 220, respectively. Significant differences to the control group were detected in the 3,4,3',4'-tetrachlorobiphenyl exposed offspring. There were increases in descent latencies in the catalepsy test and impairments of passive avoidance behavior. These behavioral effects were observed in the adult rats long after the termination of exposure when internal PCB levels were indistinguishable from those of controls. A mediation of the reported effects by alterations of dopaminergic processes or thyroid hormone levels is discussed.