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Neurotoxic effect of lead at low concentrations
Brain Research Bulletin, Vol. 55, No. 2, pp. 269 –275, 2001 Copyright © 2001 Elsevier Science Inc. Printed in the USA. All rights reserved 0361-9230/01/$–see front matter
PII S0361-9230(01)00467-1
Neurotoxic effect of lead at low concentrations O. Mameli,1* M. A. Caria,1 F. Melis,1 A. Solinas,1 C. Tavera,1 A. Ibba,2 M. Tocco,2 C. Flore2 and F. Sanna Randaccio2 1
Department of Biomedical Sciences, Division of Human Physiology, University of Sassari, Sassari, Italy; and 2 Department of Public Health, University of Cagliari, Cagliari, Italy
ABSTRACT: The effects of lead exposure at low concentrations were evaluated by studying the post-rotatory nystagmus (PRN) in two groups of rats exposed for 3 months to 50 parts per million (ppm) of sodium acetate and 50 ppm of lead acetate, respectively, in the drinking water. Only animals treated with lead acetate showed changes of the PRN parameters which were significantly related to the concentration of lead in the blood and in brain structures. The patterns of PRN responses were characterized and classified into four types: progressively inhibitory (40%), prematurely inhibitory (25%), late inhibitory (25%), and excitatory-inhibitory (10%). No alterations of the PRN parameters were observed in the animals treated with sodium acetate. The results show that exposure to lead, even at low concentrations, impairs both sensory and motor functions. The findings also point out that the vestibular system and brain stem structures which generate and control the PRN represent targets of the action of this heavy metal. Finally, the results indicate that the evaluation of the vestibulo-ocular-reflex can provide a test suited for the screening of the neurotoxic effects of lead even in the absence of clinical signs typical of lead intoxication. © 2001 Elsevier Science Inc.
tion and function of membranes causing damage, malfunction, and eventually cell death. The increase in peroxidation seems to be localized in specific cerebral areas [1], indicating that lead may not distribute uniformly within the central nervous system (CNS). Furthermore, it was recently observed that lead exerts a neurotoxic effect by interfering with the dopaminergic system, determining a reduced regulation of dopaminergic activity [13]. Because neurotransmitter release is also dependent on calcium transport, lead intoxication induces severe impairment to the dopaminergic system, acting both directly on neurotransmitter synthesis and indirectly on calcium transport. As a consequence, the signal conduction and transmission in neuronal circuits of the CNS and peripheral nervous system could be seriously impaired. Although lead toxicity has been clearly demonstrated in different systems and apparatuses, little is known on the neurotoxicity exerted by this metal at low concentrations in vivo. On this basis, in the present research we investigated the derangement of sensory and motor functions in rats exposed for 3 months at low lead concentrations. In particular, we evaluated the function of the oculomotor system because this represents a model of sensory and motor control involving not only some cortical areas, but also brain stem structures and the cerebellum. The vestibulo-ocular reflex (VOR) utilizes a circuit in which the sensory input generated by head displacement is confronted with the output of oculomotor neurons which produces the ocular movements. The best efficiency of the VOR is achieved when the relationship between the input and output, i.e., “the gain”, is near to the unit; the targeted image within the visual field would otherwise move on the retina. In other words, the feedback information deriving from the response of ocular muscles is used to adjust the gain to values corresponding to the unit, so that eye movements can be adjusted precisely until they coincide with the head movement and the image of the object stabilizes on the retina. The functional evaluation of the VOR could provide a test to assess the possible impairment of sensory and/or motor nervous functions induced by lead exposure at low concentrations.
KEY WORDS: Vestibulo-ocular-reflex, Post-rotatory-nystagmus, Heavy metals, Brainstem, Rat.
INTRODUCTION It is well known that heavy metals induce toxic effects on different systems and apparatuses [21,26]. Furthermore, because of their long half-life, heavy metals also induce accumulation phenomena, which in turn produce an exponential increase of their concentration in blood and tissues. Besides the carcinogenic effects of these compounds or their implication in chronic respiratory diseases, there is a risk that heavy metals intoxication may lead to damage of the nervous system. Among heavy metals, lead (Pb) represents the main environmental toxin. This pollutant causes well documented hematological and gastrointestinal dysfunctions, and also produces neurological impairment. A number of studies demonstrated that prolonged exposure to lead induces slower nerve conduction and an alteration of calcium homeostasis [5,15,22]. Like cadmium, nickel, and cobalt, lead interferes with ionic channels that transport calcium by calmodulin-mediated mechanisms [12,18] and compete with calcium for calmodulin-Ca2⫹ binding sites [6]. The cytotoxic effects of lead seem also related to the stimulation of peroxidative reactions occurring in the cell membrane [10,14,25]. It has been shown that, as for the cells of other tissues, lead produces in the neurons an increase of lipid peroxidation [27], which in turn determines alterations in the composi-
MATERIALS AND METHODS Long-Evans male and female rats (Charles River, Calco, Lecco, Italy), 7 weeks old at the beginning of the experiment, were used for this study. The animals were subdivided in two groups (control and exposed animals), which were stalled four per cage, at 22°C, 60 –70% humidity and with a light– dark cycle of 12 h. All rats were fed with a specific lead-free diet (Purin Basal Diet 5735”;
* Address for correspondence: Prof. Ombretta Mameli, Dept. of Biomedical Sciences: Human Physiology Division, Viale S. Pietro 43/B, 07100 Sassari, Italy. Fax: ⫹39-079-228298; E-mail: [email protected]
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270 Charles River). The experiments were conducted under authorization of the Italian Ministry of Health and institutional approval. In the control group (n ⫽ 40), sodium acetate (anhydrous, Pb ⬍ 0.0002%), at a concentration of 50 parts per million (ppm), was administered to 30 animals via the drinking water for 3 months. In the remaining animals, the basal values of Pb concentration were determined in the blood and nervous tissue samples. In the exposed group (n ⫽ 40), lead uptake occurred via the digestive tract following the administration of lead acetate (ACS Pb(CH3COO)2 䡠 3H2O), at a concentration of 50 ppm in the drinking water for 3 months. The amount of water assumed by the control and exposed animals was measured daily. The animals’ body weight was monitored weekly and a body weight curve was plotted at the end of the treatment. Vestibulo-ocular Reflex Analysis The VOR induction is known to be achieved by rotating the subject at an increasing angular velocity; when the VOR is evoked, the ocular nystagmus appears. If the rotation is abruptly stopped, the nystagmus reverses its direction and a post-rotatory nystagmus (PRN) appears. The nystagmus, which is identified by the direction of the rapid phase of the ocular movement, is generated and controlled by the vestibular nuclei, other brainstem structures, and the cerebellum [7,8,23]. Thus, the analysis of the slow phase slope, which is of vestibular origin, as well as of the rapid phase, which is of extravestibular origin, allows to investigate the efficiency of the nervous structures involved in the VOR genesis and control. For the induction of the VOR in the rat, an appropriate device was made. This included an electric motor that by means of a drive shaft produced a rotation, at a variable speed, of a platform on which the animal was fastened. Under general anesthesia (Diazepam and Ketamine, intraperitoneal, 2.5 and 37.5 mg/kg, respectively) to avoid the stress of manipulation, both the control and exposed animals were fixed in a prone position with the interaural plane parallel to the rotation plane. An appropriate head holder, fixed to the platform, allowed to immobilize the head of the animals always in the same position. The rotation was performed with an increasing velocity that reached a final angular acceleration of 180°⫺1 after 20-s rotation. The movements of the eyes on the horizontal plane of the orbit were recorded using steel needle microelectrodes (4 –5 mm in length), carefully inserted into the internal and external canthus of each eye and connected to a DC amplifier of a polygraph (Grass mod 7, 7P5C and 7DAP). The amplified signals were then conveyed to a differentiator (Grass 7P20) for the measurement of the slow phase velocity of the nystagmogram. The recording sessions were performed on both control and exposed animals, adapted to the dark, every 10 days throughout the treatment. In each session, the VOR was recorded five times, at intervals of 10 min to avoid the habituation phenomenon [9]. In each session, all responses were compared to verify the changes that appear constantly, and to select consequently the pattern characteristic of the session. In all animals the VOR was recorded before the treatment (basal conditions), thus providing individual controls. The analysis of the VOR was focused on the PRN phase, measuring its time course and onset latency, amplitude, frequency, and slow phase velocity of single jerks. At the end of the treatment, and whenever in the exposed group severe signs of VOR impairment were present, 3 ml of blood were taken from the left ventricle to evaluate lead concentration. The animals were then sacrificed with a barbiturate overdose and the brain removed to determine lead concentration in the nervous tissue. Therefore, in both groups of cases the features of PRN
MAMELI ET AL. observed at the end of the treatment were correlated in each animal to the Pb concentration in nervous tissue and blood. Analysis of Lead Concentration in the Blood and Brain To evaluate possible differences of lead distribution in various nervous structures, the metal concentration was determined in two regions, namely the telencephalon, and the brain stem-cerebellum. The analytical determination of lead concentration in these brain regions and in blood samples was carried out with the atomic absorption spectrophotometry method (Varian AA-300 Zeeman with a Techtron Zeeman GTA graphite oven, a pyrolytic graphite oven with a L’Vov platform also in pyrolytic graphite), by electrothermal atomization to perform the matrix changers. The matrix correction was based on the Zeeman effect and the signals were recorded and electronically integrated. Blood samples were diluted with a 0.2% Triton-X100 solution and Pb concentration (expressed in g/l) was calculated by comparing the absorbance of the sample with the calibration curve obtained with a standard solution of the same matrix. Brain samples, previously dried, were mineralized at wet by nitric warm attack on a heated plate. Pb concentration (g/g of dry tissue) was measured with the method of standard additions. In these experiments, the analysis of the trend of lead concentration in the blood during treatment was not performed because it was impossible to correlate it with lead concentration in the brain. Therefore, the analytical determination of blood lead concentration was performed in all animals before the beginning of the treatment (basal conditions) and at the end of the experiment. In this manner, blood and brain lead concentrations were related to the VOR features of the same animal. Ten animals of the control group were used before the beginning of the treatment as reference for the basal concentration of lead in the nervous system. These animals were sacrificed with a barbiturate overdose, the brain removed and the basal Pb values were determined in four samples per animal (the two brain hemispheres, and the two halves of the brain stem-cerebellum sectioned longitudinally). At the end of the experiment, the same procedure was applied to both groups of cases to evaluate lead concentration in the nervous tissue. Statistical analysis of data was performed in all the animals on individual PRN parameters, by means of the paired Student t-test. The same analysis was also performed for Pb concentration in blood and nervous tissue. RESULTS During lead administration and until the end of the treatment, behavioral impairment (in terms of mobility and liveliness, capacity to interact with the animals of the same cage and aggressiveness) was never observed, nor were changes detected in feeding habits or water consumption. During the treatment all animals showed a normal and gradual increase of the body weight with no statistical difference between the two groups. Control Group The analysis of the VOR demonstrated that the animals which had received 50 ppm of sodium acetate in the drinking water (n ⫽ 30) were not affected by the treatment, because they did not show significant changes of the VOR in respect to the basal conditions. The table in Fig. 1 shows the mean values (mean ⫾ standard deviation, SD) of lead concentrations, measured in basal conditions and at the end of the treatment in the blood, telencephalon, and brain stem-cerebellum of the control and exposed groups. In the control group, statistical analysis of
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FIG. 1. Lead concentrations (mean ⫾ SD) determined in basal conditions (all animals) and 80 –90 days after both sodium acetate (Control group) and lead acetate treatment (Exposed group) in blood, telencephalon and brain stem-cerebellum samples. (A) Post-rotatory nystagmus recorded in a control animal, in basal conditions (1) and during sodium acetate exposure at 47 (2), 67 (3), and 97 (4) days, respectively, from the beginning of treatment. In this and the following figures, in all traces the artifact marks the end of the rotatory stimulation. Horizontal calibration: 1 s; vertical calibration: 75 V.
Pb concentrations in these compartments did not show significant differences with the respective basal values. Figure 1A illustrates an example of a typical PRN response in a control animal. The trace A1 shows the PRN recorded in basal conditions, while traces in A2– 4 refer to the PRN of the same animal recorded after 47, 67, and 97 days of treatment, respectively. There were no significant differences in the onset latency and duration of the PRN, or in the frequency and amplitude of single jerks. The slow phase velocity of single jerks was also unaffected by sodium acetate treatment. In the representative case shown in Fig. 1A, 97 days after sodium acetate administration lead concentration was 7.7 g/l in the blood, 0.663 g/g in the brain stem-cerebellum, and 0.716 g/g in the telencephalon.
Lead-exposed Group In the animals treated with 50 ppm of lead acetate in the drinking water (n ⫽ 40), the analysis of the PRN responses showed four different patterns, designated on the basis of their characteristics as “progressively inhibitory” (40%), “prematurely inhibitory” (25%), “late inhibitory” (25%) and “excitatory-inhibitory” (10%). The table in Fig. 1 shows the mean values (⫾ SD) of lead concentrations in the exposed group, measured in basal conditions and at the end of the treatment. After 90 days of exposure to lead acetate, a significant increase of lead concentration was observed both in the blood ( p ⬍ 0.01) and nervous tissue ( p ⬍ 0.001) samples in respect to the basal conditions. Furthermore, the comparison of Pb concentration in the telencephalon and brain-
272 stem-cerebellum did not show significant differences 80 –90 days after sodium acetate administration, whereas, in the same structures, significant differences (1%) were observed 90 days after lead acetate administration in comparison to basal conditions. As for the “progressive inhibitory” response, the analysis of the PRN showed that the increase of lead concentration in the blood and nervous tissue determined a parallel and progressive worsening of the PRN parameters. In an initial phase, which corresponded to 45– 47 days of treatment, the PRN showed an impairment limited to the jerk frequency, which was significantly reduced in respect to the basal conditions ( p ⬍ 0.01). After 65– 67 days, a reduction of the amplitude of single jerks ( p ⬍ 0.001) appeared, which was followed after 80 – 82 days of treatment by a further decrease of the jerk frequency, mainly limited to the initial phase of the response (first 5 s). A slight decrease of the onset latency and a progressive reduction of the slow phase velocity were also observed in this phase of the treatment. These latter parameters were significantly altered in respect to the basal conditions ( p ⬍ 0.01). Figure 2A shows an example of this type of response; in this representative case the onset latency of PRN was 4.5 s in basal conditions (Fig. 2A1) and 5 s after 82 days of treatment (Fig. 2A4). The jerk frequency within the first 5 s decreased from 4.2 jerks/s (Fig. 2A1) to 3.2 jerks/s and the amplitude of single jerks decreased to about 70%, after only 67 days of treatment (Fig. 2A3). At the end of the treatment (82 days), the slow phase velocity of single jerks exhibited a decrease of 56% in respect to the basal values (Fig. 2A4). The lead concentration was 15 g/l in the blood, 4.21 g/g, and 4.85 g/g in the brain stem-cerebellum and telencephalon, respectively. The second pattern of response, which was defined as “prematurely inhibitory”, showed instead a marked and fast deterioration of the PRN. Impairment of the PRN was already evident 40 – 47 days after the beginning of the treatment, the most premature being the onset latency, which underwent a significant increase (150 – 170% of control value; p ⬍ 0.001). The jerk frequency, as well as the amplitude of single jerks also showed, after the same period, a significant reduction ranging from 30 – 40% ( p ⬍ 0.01), and from 50 – 60% ( p ⬍ 0.001) of the control value, respectively. A further decrease (70 – 80%) of the same parameters was observed after 82 days of lead acetate treatment. An example of this type of response is shown in Fig. 2B: the onset latency of the PRN, which was 3.4 s in basal conditions (Fig. 2B1), reached 8 s after 82 days of treatment (Fig. 2B4). At the 47th day (Fig. 2B2), the jerk frequency was reduced to 3.8 jerks/s while it was 5.2 jerks/s in basal conditions. The amplitude of single jerks and the slow phase velocity underwent a reduction of about 70% ( p ⬍ 0.01) and 80% ( p ⬍ 0.01), respectively (Fig. 2B2). In this animal at the end of the treatment (82 days), the lead concentration was 10.5 g/l in the blood, 0.941 g/g in the brain stem-cerebellum, and 1.38 g/g in the telencephalon. In the group of animals which showed the “late inhibitory” response, a clear derangement of the PRN parameters was observed only after 80 – 85 days of exposure to lead acetate. The impairment was mainly characterized by a significant increase of the onset latency (approximately 150%; p ⬍ 0.001), a reduction of the amplitude of single jerks (50%; p ⬍ 0.001), and of the total duration of the response (30%) which, however, did not reach a statistical significance, and, finally, by a decrease of the slow phase velocity (30–40%; p ⬍ 0.01). Figure 3A shows a typical example of this type of response. The onset latency, which in basal conditions was 1.4 s (Fig. 3A1), increased to 3.6 s only after 82 days of lead exposure (Fig. 3A4). The jerk frequency was only moderately reduced, but the amplitude of single jerks decreased to 53.6% in comparison with the basal value. In this animal, lead
MAMELI ET AL.
FIG. 2. (A) Post-rotary nystagmus (PRN) of a “progressive inhibitory” response, recorded in an exposed animal in basal conditions (1) and during lead acetate treatment at 47 (2), 67 (3), and 82 (4) days, respectively, from the beginning of treatment. (B) PRN of a “prematurely inhibitory” response, recorded in an animal of the exposed group, in basal conditions (A) and during lead acetate treatment at 47 (B), 67 (C), and 82 (D) days, respectively, from the beginning of treatment. Horizontal calibration: 1 s; vertical calibration: 75 V.
concentration was 12.3 g/l in the blood, 1.7 g/g in the brain stem-cerebellum, and 1.98 g/g in the telencephalon. Finally, with regard to the “excitatory-inhibitory” pattern of response, a certain analogy with the group showing a “premature inhibitory” response was observed. Even in this group, evident changes of the PRN parameters appeared after 40 – 47 days of lead treatment. However, these changes were initially of opposite sign: the onset latency of PRN was reduced (40 –50% of the basal value; p ⬍ 0.01) while the amplitude of single jerks was increased (150 –170% of the basal value; p ⬍ 0.001). The slow phase velocity also showed a parallel increase that reached 150 –160% of the control values ( p ⬍ 0.01). Around the 60th day of treatment,
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273 velocity increased up to 164% and 160% of the basal values, respectively. A gradual reduction of the excitation was detectable only after 67 days of lead exposure (Fig. 3B3); however, after 82 days, an almost complete inhibitory effect appeared (Fig. 3B4). At the end of the treatment, in this animal, lead concentration was 32 g/l in the blood, 1.56 g/g in the brain stem-cerebellum, and 1.71 g/g in the telencephalon. As for the duration of the PRN, the results demonstrated that this parameter is inconsistent. As a consequence of the treatment and independently from the type of the response, a spontaneous and long-lasting nystagmus, which followed the PRN, appeared in several animals, making the very end of the PRN difficult to assess. DISCUSSION
FIG. 3. (A) Post-rotary nystagmus (PRN) of a “late inhibitory” response, recorded in an exposed animal, in basal conditions (1) and during lead acetate treatment at 47 (2), 67 (3), and 82 (4) days, respectively, from the beginning of treatment. (B) PRN of an “excitatory-inhibitory inhibitory” response, recorded in an exposed animal, in basal conditions (1) and during lead acetate treatment at 47 (2), 67 (3), and 82 (4) days, respectively, from the beginning of treatment. Horizontal calibration: 1 s, vertical calibration: 75 V.
the PRN onset latency gradually increased ( p ⬍ 0.001), while the jerk frequency and amplitude decreased ( p ⬍ 0.01). The slow phase velocity also showed a dramatic drop (reaching 80% of the basal value; p ⬍ 0.01) and the PRN response almost disappeared 82 days after the beginning of the treatment. A typical example of this type of responses is shown in Fig. 3B. The trace in 3B1 shows that the PRN was characterized in basal conditions by an onset latency of 8.4 s, a jerk frequency of 4.6 jerks/s and a total duration of 26.6 s. In trace 3B2, 47 days after the beginning of the treatment, the onset latency was reduced to 3.5 s, the jerk frequency remained unchanged, while the jerk amplitude and the slow phase
The results show that the administration of lead acetate via the drinking water for 3 months did not induce behavioral impairment of the animals or the appearance of clinical signs of intoxication. On the contrary, in this group of cases the analysis of the VOR revealed that lead treatment induced significant changes of the PRN parameters, detectable at very low concentrations of lead in the blood and nervous tissue. In the exposed animals, the VOR impairment was strictly related to the increase of lead levels in the blood and in the brain, because in matched experimental conditions the control animals showed a normal VOR. A possible interference of the anesthesia is unlikely because control animals were subjected to the same anesthetic procedures. A number of evidences support the above consideration. Ketamine is a potent inhibitor of N-methyl-D-aspartate (NMDA) glutamate receptors (see [11] for review) which are involved in the neuronal circuits of the “neural integrator” that controls the VOR (see [24] for review). High doses of ketamine induce a failure of this “neural integrator” by increasing the VOR phase and reducing the VOR step gain [20]. Furthermore, NMDA receptors in the superior colliculus modulate the habituation of the response to visual stimulation, and ketamine administration induces a lack of habituation [4]. The latter data could also explain the appearance of a spontaneous and long-lasting nystagmus in several animals after the end of the PRN. Therefore, even though there is no direct evidence of possible interactions between lead and ketamine, altogether the above data support the hypothesis that ketamine administration should have improved the VOR features rather than impair them. Furthermore, the results of the present study demonstrate for the first time that the brain stem-cerebellum, where the neural circuits for VOR induction and control are located, is a target of lead neurotoxic action. The data also showed that it is not possible to assess a “general toxic threshold” of lead concentration in the blood and/or nervous tissue, that could allow to predict with reasonable approximation the appearance of VOR dysfunction. In the exposed group, considerable inter-individual variability in lead concentration was observed in the blood and brain at the time of clear VOR impairment. Therefore, lead action is likely to be dependent upon an “individual toxic threshold”, which is greatly variable as revealed by VOR impairment and by the high variability of the matched values of lead concentration. In particular, in the animals which showed a “premature inhibitory” PRN response, VOR modifications appeared early (after 45 days of treatment) at blood concentration of lead only slightly higher than control values and lower than those detected at the end of the treatment. Therefore, the animals showing this pattern of PRN (25%) exhibited a considerably high sensitivity of nervous structures to lead exposure, even at low concentration of this metal. Throughout the remaining period of lead exposure, a further deterioration of the PRN response was observed until it almost
274 disappeared after 3 months of treatment. It can be hypothesized than in this group the disruption of the PRN response was linearly correlated to the increase of lead concentration. On the contrary, in the group of animals which exhibited the “late inhibitory” response (25%), a deterioration of PRN parameters was observed only after 3 months of exposure to lead acetate. In these cases, the adaptive capacity of the nervous system counteracted the neurotoxic effect of lead. However, even the animals of this group showed severe impairment of the PRN parameters above a certain threshold of lead concentration in blood and brain, though consistently low. Also in the group of animals which showed the “excitatoryinhibitory” pattern (10%) the effects on PRN parameters appeared quite early (following 45 days of lead exposure) and at blood concentrations probably only slightly higher than control values. Only later, around the end of the treatment and when the lead concentration had most likely increased, the PRN was inhibited. It could be assumed that in these animals low lead concentrations in the blood and brain exerted an “irritant” action eliciting a compensatory neural mechanism, most likely responsible for the early excitatory effects. However, as lead concentration in blood and brain tissue further increased, the compensatory mechanism was no longer sufficient and severe inhibition of the PRN appeared. In the vast majority of the animals, regardless of the pattern of response, a delay of PRN onset, related to the treatment duration, was observed. This observation suggests that lead exerts a neurotoxic effect, even at low concentrations, most likely through a modification of the membrane properties of vestibular receptors and an interference with their depolarization. The well-known action of lead on the ion channel that transports calcium [12,18] could impair the depolarization mechanism of vestibular receptors. If this is the case, a temporal summation of the stimuli could overcome the reduced responsiveness of vestibular receptors and would allow the PRN response, even though with a delayed onset. However, it is difficult to assess precisely the site of lead action on the vestibular system with this experimental design, and it has been reported that a prolonged exposure to lead induces slowed nerve conduction [15]. Therefore, in the present experiments the delayed PRN onset latency could also derive from alterations in the vestibular nerve conduction. It should be considered also that the cerebellum, which exerts a complete control on both the vestibular system and oculomotor nuclei [23], could be affected by lead neurotoxicity. Purkinje cells intoxication could result in an increase of their direct inhibitory effect on the vestibular and deep cerebellar nuclei. The reduced excitatory output of the deep cerebellar nuclei could in turn be responsible for the inhibition of extrinsic ocular muscle activity, while the direct inhibition of the vestibular nuclei could explain the reduction of the PRN vestibular component. In some exposed animals, a series of spontaneous jerks, which lasted for a long time, appeared following PRN induction, making the actual PRN duration difficult to assess. This parameter does not seem, therefore, to be a reliable one. However, this observation indicates that lead also affects the inhibitory systems that in normal animals trigger the stop signal for the PRN. The anesthesia, which in some cases has been reported to induce nystagmus, cannot be responsible for this phenomenon because during the recording sessions (every 10 days) and throughout the 3 months of analysis, the control animals received the same anesthesia and never showed spontaneous jerks. As for the reduction of jerk amplitude, this could be secondary to the reduced activity of the vestibular nuclei that control the oculomotor system, as well as to the reduction of the slow phase velocity. Considering the extensive interconnections between vestibular nuclei, cerebellum, and oculomotor nuclei [2,3,16,17,23], a reduction of the intensity of vestibular and cerebellar signals to
MAMELI ET AL. oculomotor nuclei could explain the decrease of their output to the extrinsic ocular muscles and therefore account for the amplitude decrease of ocular jerks. However, it cannot be excluded that lead neurotoxicity can affect the oculomotor nuclei by a direct action on the activity of these neurons. The pattern of the “excitatory inhibitory” responses supports the possibility that in some subjects lead intoxication may induce a dose-dependent biphasic effect, so that at low concentrations lead may increase the excitability of VOR neuronal circuits whereas at higher concentrations may induce a generalized inhibition, analogous to that observed for the other pattern of response. Finally, the results showed a significant difference of lead distribution in the tested nervous structures with a higher concentration in the telencephalon, in which the concentration of the heavy metal was about twice that of the brain stem-cerebellum. It is known that lead intoxication impairs the cognitive functions [19,28]. However, since significant disruption of the VOR was observed without concomitant behavioral effects, the brain stemcerebellum could represent a target of lead intoxication more sensitive than other brain structures. On the basis of the present results, and considering that VOR induction is a non-invasive method, of low cost, and easy to apply, its use could provide a test for screening the exposed population with the aim of identifying individual toxic thresholds before the appearance of clinical signs of lead intoxication. VOR induction with caloric stimulation of the labyrinth could provide an equally effective method. The caloric stimulation of the labyrinth generates an endolymphatic current analogous to that obtained by the natural method of rotatory stimulation, could be applied at lower cost, in less time and on a broader population. In conclusion, the results of the present experiment demonstrate for the first time that (1) lead exerts a neurotoxic effect, resulting in sensory and motor function impairment, even at low concentrations; (2) the brain stem and the cerebellum are targets of the action of this heavy metal; and (3) the analysis of the VOR is suitable to assess the neurotoxic effects of lead long before the appearance of clinical signs of intoxication. ACKNOWLEDGEMENTS
We wish to thank Mr. Giancarlo Sanna, Mr. Andrea Monti and Mr. Candido Tavera for their technical assistance.
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Legano, L. A.; Kruger, H. A.; Lim, S. W.; Barasch, S.; Au, L.; Courtlandt, C. D. Low-level lead exposure and cognitive development in early childhood. J. Dev. Behav. Pediatr. 20:425– 431; 1999. Mettens, P.; Godaux, E.; Cheron, G. Effects of ketamine on ocular movements of the cat. J. Vestib. Res. 1:325–338; 1990 –91. Nordberg, M. General aspects of cadmium: Transport, uptake and metabolism by the kidney. Environ. Health Perspect. 54:13–20; 1984. Orrenius, S.; Burkitt, M. J.; Kass, G. E.; Dypbukt, J. M.; Nicotera P., Calcium ions and oxidative cell injury. Ann. Neurol. 32(suppl.):33– 42; 1992. Pompeiano, O. Cerebello-vestibular interrelations. In: Kornhuber, H. H., ed. Handbook of sensory physiology, vol. VI/1. Vestibular system. Berlin, Heidelberg, New York: Springer-Verlag; 1974:417– 476. Priesol, A. J.; Jones, G. E.; Tomlinson, R. D.; Broussard, D. M. Frequency-dependent effects of glutamate antagonists on the vestibulo-ocular reflex of the cat. Brain Res. 857:252–264; 2000. Ramstoeck, E. R.; Hoekstra, W. G.; Ganther, H. E. Trialkyllead metabolism and lipid peroxidation in vivo in vitamin E-and seleniumdeficient rats, as measured by ethane production. Toxicol Appl. Pharmacol. 54:251–257; 1980. Revis, N. A possible mechanism for cadmium-induced hypertension in rats. Life Sci. 22:479 – 487; 1978. Shafiq-Ur-Rehman, S. Lead-induced regional lipid peroxidation in brain. Toxicol. Lett. 21:333–337; 1984. Soong, W. T.; Chao, K. Y.; Jang, C. S.; Wang, J. D. Long-term effect of increased lead absorption on intelligence of children. Arch. Environ. Health 54:297–301; 1999.
PII S0361-9230(01)00467-1
Neurotoxic effect of lead at low concentrations O. Mameli,1* M. A. Caria,1 F. Melis,1 A. Solinas,1 C. Tavera,1 A. Ibba,2 M. Tocco,2 C. Flore2 and F. Sanna Randaccio2 1
Department of Biomedical Sciences, Division of Human Physiology, University of Sassari, Sassari, Italy; and 2 Department of Public Health, University of Cagliari, Cagliari, Italy
ABSTRACT: The effects of lead exposure at low concentrations were evaluated by studying the post-rotatory nystagmus (PRN) in two groups of rats exposed for 3 months to 50 parts per million (ppm) of sodium acetate and 50 ppm of lead acetate, respectively, in the drinking water. Only animals treated with lead acetate showed changes of the PRN parameters which were significantly related to the concentration of lead in the blood and in brain structures. The patterns of PRN responses were characterized and classified into four types: progressively inhibitory (40%), prematurely inhibitory (25%), late inhibitory (25%), and excitatory-inhibitory (10%). No alterations of the PRN parameters were observed in the animals treated with sodium acetate. The results show that exposure to lead, even at low concentrations, impairs both sensory and motor functions. The findings also point out that the vestibular system and brain stem structures which generate and control the PRN represent targets of the action of this heavy metal. Finally, the results indicate that the evaluation of the vestibulo-ocular-reflex can provide a test suited for the screening of the neurotoxic effects of lead even in the absence of clinical signs typical of lead intoxication. © 2001 Elsevier Science Inc.
tion and function of membranes causing damage, malfunction, and eventually cell death. The increase in peroxidation seems to be localized in specific cerebral areas [1], indicating that lead may not distribute uniformly within the central nervous system (CNS). Furthermore, it was recently observed that lead exerts a neurotoxic effect by interfering with the dopaminergic system, determining a reduced regulation of dopaminergic activity [13]. Because neurotransmitter release is also dependent on calcium transport, lead intoxication induces severe impairment to the dopaminergic system, acting both directly on neurotransmitter synthesis and indirectly on calcium transport. As a consequence, the signal conduction and transmission in neuronal circuits of the CNS and peripheral nervous system could be seriously impaired. Although lead toxicity has been clearly demonstrated in different systems and apparatuses, little is known on the neurotoxicity exerted by this metal at low concentrations in vivo. On this basis, in the present research we investigated the derangement of sensory and motor functions in rats exposed for 3 months at low lead concentrations. In particular, we evaluated the function of the oculomotor system because this represents a model of sensory and motor control involving not only some cortical areas, but also brain stem structures and the cerebellum. The vestibulo-ocular reflex (VOR) utilizes a circuit in which the sensory input generated by head displacement is confronted with the output of oculomotor neurons which produces the ocular movements. The best efficiency of the VOR is achieved when the relationship between the input and output, i.e., “the gain”, is near to the unit; the targeted image within the visual field would otherwise move on the retina. In other words, the feedback information deriving from the response of ocular muscles is used to adjust the gain to values corresponding to the unit, so that eye movements can be adjusted precisely until they coincide with the head movement and the image of the object stabilizes on the retina. The functional evaluation of the VOR could provide a test to assess the possible impairment of sensory and/or motor nervous functions induced by lead exposure at low concentrations.
KEY WORDS: Vestibulo-ocular-reflex, Post-rotatory-nystagmus, Heavy metals, Brainstem, Rat.
INTRODUCTION It is well known that heavy metals induce toxic effects on different systems and apparatuses [21,26]. Furthermore, because of their long half-life, heavy metals also induce accumulation phenomena, which in turn produce an exponential increase of their concentration in blood and tissues. Besides the carcinogenic effects of these compounds or their implication in chronic respiratory diseases, there is a risk that heavy metals intoxication may lead to damage of the nervous system. Among heavy metals, lead (Pb) represents the main environmental toxin. This pollutant causes well documented hematological and gastrointestinal dysfunctions, and also produces neurological impairment. A number of studies demonstrated that prolonged exposure to lead induces slower nerve conduction and an alteration of calcium homeostasis [5,15,22]. Like cadmium, nickel, and cobalt, lead interferes with ionic channels that transport calcium by calmodulin-mediated mechanisms [12,18] and compete with calcium for calmodulin-Ca2⫹ binding sites [6]. The cytotoxic effects of lead seem also related to the stimulation of peroxidative reactions occurring in the cell membrane [10,14,25]. It has been shown that, as for the cells of other tissues, lead produces in the neurons an increase of lipid peroxidation [27], which in turn determines alterations in the composi-
MATERIALS AND METHODS Long-Evans male and female rats (Charles River, Calco, Lecco, Italy), 7 weeks old at the beginning of the experiment, were used for this study. The animals were subdivided in two groups (control and exposed animals), which were stalled four per cage, at 22°C, 60 –70% humidity and with a light– dark cycle of 12 h. All rats were fed with a specific lead-free diet (Purin Basal Diet 5735”;
* Address for correspondence: Prof. Ombretta Mameli, Dept. of Biomedical Sciences: Human Physiology Division, Viale S. Pietro 43/B, 07100 Sassari, Italy. Fax: ⫹39-079-228298; E-mail: [email protected]
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270 Charles River). The experiments were conducted under authorization of the Italian Ministry of Health and institutional approval. In the control group (n ⫽ 40), sodium acetate (anhydrous, Pb ⬍ 0.0002%), at a concentration of 50 parts per million (ppm), was administered to 30 animals via the drinking water for 3 months. In the remaining animals, the basal values of Pb concentration were determined in the blood and nervous tissue samples. In the exposed group (n ⫽ 40), lead uptake occurred via the digestive tract following the administration of lead acetate (ACS Pb(CH3COO)2 䡠 3H2O), at a concentration of 50 ppm in the drinking water for 3 months. The amount of water assumed by the control and exposed animals was measured daily. The animals’ body weight was monitored weekly and a body weight curve was plotted at the end of the treatment. Vestibulo-ocular Reflex Analysis The VOR induction is known to be achieved by rotating the subject at an increasing angular velocity; when the VOR is evoked, the ocular nystagmus appears. If the rotation is abruptly stopped, the nystagmus reverses its direction and a post-rotatory nystagmus (PRN) appears. The nystagmus, which is identified by the direction of the rapid phase of the ocular movement, is generated and controlled by the vestibular nuclei, other brainstem structures, and the cerebellum [7,8,23]. Thus, the analysis of the slow phase slope, which is of vestibular origin, as well as of the rapid phase, which is of extravestibular origin, allows to investigate the efficiency of the nervous structures involved in the VOR genesis and control. For the induction of the VOR in the rat, an appropriate device was made. This included an electric motor that by means of a drive shaft produced a rotation, at a variable speed, of a platform on which the animal was fastened. Under general anesthesia (Diazepam and Ketamine, intraperitoneal, 2.5 and 37.5 mg/kg, respectively) to avoid the stress of manipulation, both the control and exposed animals were fixed in a prone position with the interaural plane parallel to the rotation plane. An appropriate head holder, fixed to the platform, allowed to immobilize the head of the animals always in the same position. The rotation was performed with an increasing velocity that reached a final angular acceleration of 180°⫺1 after 20-s rotation. The movements of the eyes on the horizontal plane of the orbit were recorded using steel needle microelectrodes (4 –5 mm in length), carefully inserted into the internal and external canthus of each eye and connected to a DC amplifier of a polygraph (Grass mod 7, 7P5C and 7DAP). The amplified signals were then conveyed to a differentiator (Grass 7P20) for the measurement of the slow phase velocity of the nystagmogram. The recording sessions were performed on both control and exposed animals, adapted to the dark, every 10 days throughout the treatment. In each session, the VOR was recorded five times, at intervals of 10 min to avoid the habituation phenomenon [9]. In each session, all responses were compared to verify the changes that appear constantly, and to select consequently the pattern characteristic of the session. In all animals the VOR was recorded before the treatment (basal conditions), thus providing individual controls. The analysis of the VOR was focused on the PRN phase, measuring its time course and onset latency, amplitude, frequency, and slow phase velocity of single jerks. At the end of the treatment, and whenever in the exposed group severe signs of VOR impairment were present, 3 ml of blood were taken from the left ventricle to evaluate lead concentration. The animals were then sacrificed with a barbiturate overdose and the brain removed to determine lead concentration in the nervous tissue. Therefore, in both groups of cases the features of PRN
MAMELI ET AL. observed at the end of the treatment were correlated in each animal to the Pb concentration in nervous tissue and blood. Analysis of Lead Concentration in the Blood and Brain To evaluate possible differences of lead distribution in various nervous structures, the metal concentration was determined in two regions, namely the telencephalon, and the brain stem-cerebellum. The analytical determination of lead concentration in these brain regions and in blood samples was carried out with the atomic absorption spectrophotometry method (Varian AA-300 Zeeman with a Techtron Zeeman GTA graphite oven, a pyrolytic graphite oven with a L’Vov platform also in pyrolytic graphite), by electrothermal atomization to perform the matrix changers. The matrix correction was based on the Zeeman effect and the signals were recorded and electronically integrated. Blood samples were diluted with a 0.2% Triton-X100 solution and Pb concentration (expressed in g/l) was calculated by comparing the absorbance of the sample with the calibration curve obtained with a standard solution of the same matrix. Brain samples, previously dried, were mineralized at wet by nitric warm attack on a heated plate. Pb concentration (g/g of dry tissue) was measured with the method of standard additions. In these experiments, the analysis of the trend of lead concentration in the blood during treatment was not performed because it was impossible to correlate it with lead concentration in the brain. Therefore, the analytical determination of blood lead concentration was performed in all animals before the beginning of the treatment (basal conditions) and at the end of the experiment. In this manner, blood and brain lead concentrations were related to the VOR features of the same animal. Ten animals of the control group were used before the beginning of the treatment as reference for the basal concentration of lead in the nervous system. These animals were sacrificed with a barbiturate overdose, the brain removed and the basal Pb values were determined in four samples per animal (the two brain hemispheres, and the two halves of the brain stem-cerebellum sectioned longitudinally). At the end of the experiment, the same procedure was applied to both groups of cases to evaluate lead concentration in the nervous tissue. Statistical analysis of data was performed in all the animals on individual PRN parameters, by means of the paired Student t-test. The same analysis was also performed for Pb concentration in blood and nervous tissue. RESULTS During lead administration and until the end of the treatment, behavioral impairment (in terms of mobility and liveliness, capacity to interact with the animals of the same cage and aggressiveness) was never observed, nor were changes detected in feeding habits or water consumption. During the treatment all animals showed a normal and gradual increase of the body weight with no statistical difference between the two groups. Control Group The analysis of the VOR demonstrated that the animals which had received 50 ppm of sodium acetate in the drinking water (n ⫽ 30) were not affected by the treatment, because they did not show significant changes of the VOR in respect to the basal conditions. The table in Fig. 1 shows the mean values (mean ⫾ standard deviation, SD) of lead concentrations, measured in basal conditions and at the end of the treatment in the blood, telencephalon, and brain stem-cerebellum of the control and exposed groups. In the control group, statistical analysis of
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FIG. 1. Lead concentrations (mean ⫾ SD) determined in basal conditions (all animals) and 80 –90 days after both sodium acetate (Control group) and lead acetate treatment (Exposed group) in blood, telencephalon and brain stem-cerebellum samples. (A) Post-rotatory nystagmus recorded in a control animal, in basal conditions (1) and during sodium acetate exposure at 47 (2), 67 (3), and 97 (4) days, respectively, from the beginning of treatment. In this and the following figures, in all traces the artifact marks the end of the rotatory stimulation. Horizontal calibration: 1 s; vertical calibration: 75 V.
Pb concentrations in these compartments did not show significant differences with the respective basal values. Figure 1A illustrates an example of a typical PRN response in a control animal. The trace A1 shows the PRN recorded in basal conditions, while traces in A2– 4 refer to the PRN of the same animal recorded after 47, 67, and 97 days of treatment, respectively. There were no significant differences in the onset latency and duration of the PRN, or in the frequency and amplitude of single jerks. The slow phase velocity of single jerks was also unaffected by sodium acetate treatment. In the representative case shown in Fig. 1A, 97 days after sodium acetate administration lead concentration was 7.7 g/l in the blood, 0.663 g/g in the brain stem-cerebellum, and 0.716 g/g in the telencephalon.
Lead-exposed Group In the animals treated with 50 ppm of lead acetate in the drinking water (n ⫽ 40), the analysis of the PRN responses showed four different patterns, designated on the basis of their characteristics as “progressively inhibitory” (40%), “prematurely inhibitory” (25%), “late inhibitory” (25%) and “excitatory-inhibitory” (10%). The table in Fig. 1 shows the mean values (⫾ SD) of lead concentrations in the exposed group, measured in basal conditions and at the end of the treatment. After 90 days of exposure to lead acetate, a significant increase of lead concentration was observed both in the blood ( p ⬍ 0.01) and nervous tissue ( p ⬍ 0.001) samples in respect to the basal conditions. Furthermore, the comparison of Pb concentration in the telencephalon and brain-
272 stem-cerebellum did not show significant differences 80 –90 days after sodium acetate administration, whereas, in the same structures, significant differences (1%) were observed 90 days after lead acetate administration in comparison to basal conditions. As for the “progressive inhibitory” response, the analysis of the PRN showed that the increase of lead concentration in the blood and nervous tissue determined a parallel and progressive worsening of the PRN parameters. In an initial phase, which corresponded to 45– 47 days of treatment, the PRN showed an impairment limited to the jerk frequency, which was significantly reduced in respect to the basal conditions ( p ⬍ 0.01). After 65– 67 days, a reduction of the amplitude of single jerks ( p ⬍ 0.001) appeared, which was followed after 80 – 82 days of treatment by a further decrease of the jerk frequency, mainly limited to the initial phase of the response (first 5 s). A slight decrease of the onset latency and a progressive reduction of the slow phase velocity were also observed in this phase of the treatment. These latter parameters were significantly altered in respect to the basal conditions ( p ⬍ 0.01). Figure 2A shows an example of this type of response; in this representative case the onset latency of PRN was 4.5 s in basal conditions (Fig. 2A1) and 5 s after 82 days of treatment (Fig. 2A4). The jerk frequency within the first 5 s decreased from 4.2 jerks/s (Fig. 2A1) to 3.2 jerks/s and the amplitude of single jerks decreased to about 70%, after only 67 days of treatment (Fig. 2A3). At the end of the treatment (82 days), the slow phase velocity of single jerks exhibited a decrease of 56% in respect to the basal values (Fig. 2A4). The lead concentration was 15 g/l in the blood, 4.21 g/g, and 4.85 g/g in the brain stem-cerebellum and telencephalon, respectively. The second pattern of response, which was defined as “prematurely inhibitory”, showed instead a marked and fast deterioration of the PRN. Impairment of the PRN was already evident 40 – 47 days after the beginning of the treatment, the most premature being the onset latency, which underwent a significant increase (150 – 170% of control value; p ⬍ 0.001). The jerk frequency, as well as the amplitude of single jerks also showed, after the same period, a significant reduction ranging from 30 – 40% ( p ⬍ 0.01), and from 50 – 60% ( p ⬍ 0.001) of the control value, respectively. A further decrease (70 – 80%) of the same parameters was observed after 82 days of lead acetate treatment. An example of this type of response is shown in Fig. 2B: the onset latency of the PRN, which was 3.4 s in basal conditions (Fig. 2B1), reached 8 s after 82 days of treatment (Fig. 2B4). At the 47th day (Fig. 2B2), the jerk frequency was reduced to 3.8 jerks/s while it was 5.2 jerks/s in basal conditions. The amplitude of single jerks and the slow phase velocity underwent a reduction of about 70% ( p ⬍ 0.01) and 80% ( p ⬍ 0.01), respectively (Fig. 2B2). In this animal at the end of the treatment (82 days), the lead concentration was 10.5 g/l in the blood, 0.941 g/g in the brain stem-cerebellum, and 1.38 g/g in the telencephalon. In the group of animals which showed the “late inhibitory” response, a clear derangement of the PRN parameters was observed only after 80 – 85 days of exposure to lead acetate. The impairment was mainly characterized by a significant increase of the onset latency (approximately 150%; p ⬍ 0.001), a reduction of the amplitude of single jerks (50%; p ⬍ 0.001), and of the total duration of the response (30%) which, however, did not reach a statistical significance, and, finally, by a decrease of the slow phase velocity (30–40%; p ⬍ 0.01). Figure 3A shows a typical example of this type of response. The onset latency, which in basal conditions was 1.4 s (Fig. 3A1), increased to 3.6 s only after 82 days of lead exposure (Fig. 3A4). The jerk frequency was only moderately reduced, but the amplitude of single jerks decreased to 53.6% in comparison with the basal value. In this animal, lead
MAMELI ET AL.
FIG. 2. (A) Post-rotary nystagmus (PRN) of a “progressive inhibitory” response, recorded in an exposed animal in basal conditions (1) and during lead acetate treatment at 47 (2), 67 (3), and 82 (4) days, respectively, from the beginning of treatment. (B) PRN of a “prematurely inhibitory” response, recorded in an animal of the exposed group, in basal conditions (A) and during lead acetate treatment at 47 (B), 67 (C), and 82 (D) days, respectively, from the beginning of treatment. Horizontal calibration: 1 s; vertical calibration: 75 V.
concentration was 12.3 g/l in the blood, 1.7 g/g in the brain stem-cerebellum, and 1.98 g/g in the telencephalon. Finally, with regard to the “excitatory-inhibitory” pattern of response, a certain analogy with the group showing a “premature inhibitory” response was observed. Even in this group, evident changes of the PRN parameters appeared after 40 – 47 days of lead treatment. However, these changes were initially of opposite sign: the onset latency of PRN was reduced (40 –50% of the basal value; p ⬍ 0.01) while the amplitude of single jerks was increased (150 –170% of the basal value; p ⬍ 0.001). The slow phase velocity also showed a parallel increase that reached 150 –160% of the control values ( p ⬍ 0.01). Around the 60th day of treatment,
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273 velocity increased up to 164% and 160% of the basal values, respectively. A gradual reduction of the excitation was detectable only after 67 days of lead exposure (Fig. 3B3); however, after 82 days, an almost complete inhibitory effect appeared (Fig. 3B4). At the end of the treatment, in this animal, lead concentration was 32 g/l in the blood, 1.56 g/g in the brain stem-cerebellum, and 1.71 g/g in the telencephalon. As for the duration of the PRN, the results demonstrated that this parameter is inconsistent. As a consequence of the treatment and independently from the type of the response, a spontaneous and long-lasting nystagmus, which followed the PRN, appeared in several animals, making the very end of the PRN difficult to assess. DISCUSSION
FIG. 3. (A) Post-rotary nystagmus (PRN) of a “late inhibitory” response, recorded in an exposed animal, in basal conditions (1) and during lead acetate treatment at 47 (2), 67 (3), and 82 (4) days, respectively, from the beginning of treatment. (B) PRN of an “excitatory-inhibitory inhibitory” response, recorded in an exposed animal, in basal conditions (1) and during lead acetate treatment at 47 (2), 67 (3), and 82 (4) days, respectively, from the beginning of treatment. Horizontal calibration: 1 s, vertical calibration: 75 V.
the PRN onset latency gradually increased ( p ⬍ 0.001), while the jerk frequency and amplitude decreased ( p ⬍ 0.01). The slow phase velocity also showed a dramatic drop (reaching 80% of the basal value; p ⬍ 0.01) and the PRN response almost disappeared 82 days after the beginning of the treatment. A typical example of this type of responses is shown in Fig. 3B. The trace in 3B1 shows that the PRN was characterized in basal conditions by an onset latency of 8.4 s, a jerk frequency of 4.6 jerks/s and a total duration of 26.6 s. In trace 3B2, 47 days after the beginning of the treatment, the onset latency was reduced to 3.5 s, the jerk frequency remained unchanged, while the jerk amplitude and the slow phase
The results show that the administration of lead acetate via the drinking water for 3 months did not induce behavioral impairment of the animals or the appearance of clinical signs of intoxication. On the contrary, in this group of cases the analysis of the VOR revealed that lead treatment induced significant changes of the PRN parameters, detectable at very low concentrations of lead in the blood and nervous tissue. In the exposed animals, the VOR impairment was strictly related to the increase of lead levels in the blood and in the brain, because in matched experimental conditions the control animals showed a normal VOR. A possible interference of the anesthesia is unlikely because control animals were subjected to the same anesthetic procedures. A number of evidences support the above consideration. Ketamine is a potent inhibitor of N-methyl-D-aspartate (NMDA) glutamate receptors (see [11] for review) which are involved in the neuronal circuits of the “neural integrator” that controls the VOR (see [24] for review). High doses of ketamine induce a failure of this “neural integrator” by increasing the VOR phase and reducing the VOR step gain [20]. Furthermore, NMDA receptors in the superior colliculus modulate the habituation of the response to visual stimulation, and ketamine administration induces a lack of habituation [4]. The latter data could also explain the appearance of a spontaneous and long-lasting nystagmus in several animals after the end of the PRN. Therefore, even though there is no direct evidence of possible interactions between lead and ketamine, altogether the above data support the hypothesis that ketamine administration should have improved the VOR features rather than impair them. Furthermore, the results of the present study demonstrate for the first time that the brain stem-cerebellum, where the neural circuits for VOR induction and control are located, is a target of lead neurotoxic action. The data also showed that it is not possible to assess a “general toxic threshold” of lead concentration in the blood and/or nervous tissue, that could allow to predict with reasonable approximation the appearance of VOR dysfunction. In the exposed group, considerable inter-individual variability in lead concentration was observed in the blood and brain at the time of clear VOR impairment. Therefore, lead action is likely to be dependent upon an “individual toxic threshold”, which is greatly variable as revealed by VOR impairment and by the high variability of the matched values of lead concentration. In particular, in the animals which showed a “premature inhibitory” PRN response, VOR modifications appeared early (after 45 days of treatment) at blood concentration of lead only slightly higher than control values and lower than those detected at the end of the treatment. Therefore, the animals showing this pattern of PRN (25%) exhibited a considerably high sensitivity of nervous structures to lead exposure, even at low concentration of this metal. Throughout the remaining period of lead exposure, a further deterioration of the PRN response was observed until it almost
274 disappeared after 3 months of treatment. It can be hypothesized than in this group the disruption of the PRN response was linearly correlated to the increase of lead concentration. On the contrary, in the group of animals which exhibited the “late inhibitory” response (25%), a deterioration of PRN parameters was observed only after 3 months of exposure to lead acetate. In these cases, the adaptive capacity of the nervous system counteracted the neurotoxic effect of lead. However, even the animals of this group showed severe impairment of the PRN parameters above a certain threshold of lead concentration in blood and brain, though consistently low. Also in the group of animals which showed the “excitatoryinhibitory” pattern (10%) the effects on PRN parameters appeared quite early (following 45 days of lead exposure) and at blood concentrations probably only slightly higher than control values. Only later, around the end of the treatment and when the lead concentration had most likely increased, the PRN was inhibited. It could be assumed that in these animals low lead concentrations in the blood and brain exerted an “irritant” action eliciting a compensatory neural mechanism, most likely responsible for the early excitatory effects. However, as lead concentration in blood and brain tissue further increased, the compensatory mechanism was no longer sufficient and severe inhibition of the PRN appeared. In the vast majority of the animals, regardless of the pattern of response, a delay of PRN onset, related to the treatment duration, was observed. This observation suggests that lead exerts a neurotoxic effect, even at low concentrations, most likely through a modification of the membrane properties of vestibular receptors and an interference with their depolarization. The well-known action of lead on the ion channel that transports calcium [12,18] could impair the depolarization mechanism of vestibular receptors. If this is the case, a temporal summation of the stimuli could overcome the reduced responsiveness of vestibular receptors and would allow the PRN response, even though with a delayed onset. However, it is difficult to assess precisely the site of lead action on the vestibular system with this experimental design, and it has been reported that a prolonged exposure to lead induces slowed nerve conduction [15]. Therefore, in the present experiments the delayed PRN onset latency could also derive from alterations in the vestibular nerve conduction. It should be considered also that the cerebellum, which exerts a complete control on both the vestibular system and oculomotor nuclei [23], could be affected by lead neurotoxicity. Purkinje cells intoxication could result in an increase of their direct inhibitory effect on the vestibular and deep cerebellar nuclei. The reduced excitatory output of the deep cerebellar nuclei could in turn be responsible for the inhibition of extrinsic ocular muscle activity, while the direct inhibition of the vestibular nuclei could explain the reduction of the PRN vestibular component. In some exposed animals, a series of spontaneous jerks, which lasted for a long time, appeared following PRN induction, making the actual PRN duration difficult to assess. This parameter does not seem, therefore, to be a reliable one. However, this observation indicates that lead also affects the inhibitory systems that in normal animals trigger the stop signal for the PRN. The anesthesia, which in some cases has been reported to induce nystagmus, cannot be responsible for this phenomenon because during the recording sessions (every 10 days) and throughout the 3 months of analysis, the control animals received the same anesthesia and never showed spontaneous jerks. As for the reduction of jerk amplitude, this could be secondary to the reduced activity of the vestibular nuclei that control the oculomotor system, as well as to the reduction of the slow phase velocity. Considering the extensive interconnections between vestibular nuclei, cerebellum, and oculomotor nuclei [2,3,16,17,23], a reduction of the intensity of vestibular and cerebellar signals to
MAMELI ET AL. oculomotor nuclei could explain the decrease of their output to the extrinsic ocular muscles and therefore account for the amplitude decrease of ocular jerks. However, it cannot be excluded that lead neurotoxicity can affect the oculomotor nuclei by a direct action on the activity of these neurons. The pattern of the “excitatory inhibitory” responses supports the possibility that in some subjects lead intoxication may induce a dose-dependent biphasic effect, so that at low concentrations lead may increase the excitability of VOR neuronal circuits whereas at higher concentrations may induce a generalized inhibition, analogous to that observed for the other pattern of response. Finally, the results showed a significant difference of lead distribution in the tested nervous structures with a higher concentration in the telencephalon, in which the concentration of the heavy metal was about twice that of the brain stem-cerebellum. It is known that lead intoxication impairs the cognitive functions [19,28]. However, since significant disruption of the VOR was observed without concomitant behavioral effects, the brain stemcerebellum could represent a target of lead intoxication more sensitive than other brain structures. On the basis of the present results, and considering that VOR induction is a non-invasive method, of low cost, and easy to apply, its use could provide a test for screening the exposed population with the aim of identifying individual toxic thresholds before the appearance of clinical signs of lead intoxication. VOR induction with caloric stimulation of the labyrinth could provide an equally effective method. The caloric stimulation of the labyrinth generates an endolymphatic current analogous to that obtained by the natural method of rotatory stimulation, could be applied at lower cost, in less time and on a broader population. In conclusion, the results of the present experiment demonstrate for the first time that (1) lead exerts a neurotoxic effect, resulting in sensory and motor function impairment, even at low concentrations; (2) the brain stem and the cerebellum are targets of the action of this heavy metal; and (3) the analysis of the VOR is suitable to assess the neurotoxic effects of lead long before the appearance of clinical signs of intoxication. ACKNOWLEDGEMENTS
We wish to thank Mr. Giancarlo Sanna, Mr. Andrea Monti and Mr. Candido Tavera for their technical assistance.
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