Neurotoxicology and Teratology, Vol. 19, No. 6, pp. 489497, 1997 Copyright 1997 Elsevier Science Inc.

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Effects of LeadArsenic Combined Exposure on Central Monoaminergic Systems

J. J. MEJA,* F. DAZ-BARRIGA,* J. CALDERN,* C. ROS AND M. E. JIMNEZ-CAPDEVILLE * Laboratorio de Toxicologa Ambiental, Departamento de Biologa Celular, Facultad de Medicina, Universidad Autnoma de San Luis Potos, Mxico Departamento de Neuroqumica, Instituto Nacional de Neurologa y Neurociruga Manuel Velasco Surez, Mxico D.F., Mxico Departamento de Bioqumica, Facultad de Medicina, Universidad Autnoma de San Luis Potos, Av. Venustiano Carranza 2405, 78210 San Luis Potos S.L.P. Mxico Received 10 December 1996; Accepted 18 May 1997
MEJA, J. J., F. DAZ-BARRIGA, J. CALDERN, C. ROS AND M. E. JIMNEZ-CAPDEVILLE. Effects of lead arsenic combined exposure on central monoaminergic systems. NEUROTOXICOL TERATOL 19(6) 489497, 1997.Lead acetate (116 mg/kg/day), arsenic (11 or 13.8 mg/kg/day as sodium arsenite), a leadarsenic mixture or vehicle were administered to adult mice through gastric intubation during 14 days. Then, the regional content of norepinephrine (NE), dopamine (DA), serotonin (5-HT), 3,4 dihydroxyphenyl-acetic acid (DOPAC), 5-hydroxyindole-3-acetic acid (5-HIAA), arsenic, and lead were quantified. Compared with the accumulation after single element exposures, the mixture elicited a higher accumulation of lead and a lower arsenic accumulation in the brain. Compared to controls, lead induced only an augmentation of DOPAC (200%) in the hypothalamus. By contrast, the mixture provoked increases of DOPAC in the hypothalamus (250%), DA and 5-HIAA in the striatum (67 and 187%, respectively) and NE decreased in the hypothalamus (45%). Although these alterations were similar to those produced by arsenic alone, the mixture provoked a 38% decrease of NE in the hippocampus and increases of 5-HT in midbrain and frontal cortex (100 and 90%, respectively) over control values, alterations that were not elicited by either metal alone. These results demonstrate an interaction arsenic/lead on the central monaminergic systems of the adult mouse. 1997 Elsevier Science Inc. Lead Arsenic Catecholamines Serotonin Chemical mixtures

ARSENIC and lead are naturally occurring elements that are widely distributed in the environment. All humans are exposed to low levels of these elements; however, above-average levels of coexposure to lead and arsenic are associated with the smelting of copper (17). Both elements are neurotoxic. There is evidence that exposure during childhood to relatively low lead levels (10 g/dl of blood) produces cognitive deficits (1,9). Animal studies have contributed insight into the impact of lead exposure on the nervous system. Over a decade ago, several studies in animals suggested that the metabolism of biogenic amines is influenced by lead (12,18,40). The effects of lead at different levels of biogenic amine neurochemistry, namely, metabolism, uptake, synthesis, storage, and release, have been studied in

animals, because of the role of these systems in the control of important behavioral functions. Arsenic may affect the central and peripheral nervous systems. In the central nervous system an acute exposure (1 mg/kg/ day, in humans) may cause encephalopathy (2,13). Intermediate and chronic duration exposures to lower levels (0.02 0.5 mg/kg/day) are characterized by peripheral neuropathy (22, 57) involving sensory and motor pathways, which show distal axon degeneration and demyelination (25). We have previously reported that children exposed to arsenic (600 mg/kg of soil, 1000 mg/kg of dust) and other metals in San Luis Potos show symptoms of central nervous system alterations, such as sleep disturbances, abnormal abilities or performance, and altered latency of auditive-evoked potentials (17,44).

To whom requests for reprints should be addressed. Dr. M. E. Jimnez-Capdeville, Departamento de Bioqumica, Facultad de Medicina, Av. Venustiano Carranza 2405, 78210 San Luis Potos S.L.P., Mxico.

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490 At present there is an increasing interest in studying the effects of exposure to combinations of chemicals, because people are simultaneously exposed to a variety of toxic agents in the environment. For instance, recent investigations about the effects of lead exposure in humans, especially children, have tried to correlate neurobehavioral abnormalities with blood lead levels; however, one of the major shortcomings of those studies is that the subjects were simultaneously exposed to several neurotoxicants (4,14,50). In mining areas or in the vicinity of copper smelters, arsenic is often present together with lead. The present study was undertaken to investigate the effects of arsenic exposure on central monoaminergic systems and to asses to what extent the reported effects of lead upon these systems are affected by the presence of arsenic. After 14 days of oral administration of arsenic, lead or the leadarsenic mixture, the levels of three monoamines: dopamine, norepinephrine, and serotonin (DA, NE, and 5-HT) and of two metabolites: 3,4-dihydroxyphenylacetic acid (DOPAC) and 5-hydroxyindole-3-acetic acid (5-HIAA), as well as the concentrations of lead and arsenic were assesed in specific regions of the brain.
METHOD

MEJA ET AL. homogenized by sonication in ice-cold 0.05 M perchloric acid containing 1% of sodium metabisulfite (Sigma Chemical Co., St. Louis, MO). The homogenates were centrifuged and the pellets discarded. Supernatants were stored at 20C until analysis for monoamine content by HPLC coupled with electrochemical detection, using established procedures (45,48). A chromatographic column from Alltech Associates Inc. (Deerfield, IL) packed with adsorbosphere (3 mm particle size, 100 4.8 mm) was used. The isocratic mobile phase was a phosphate buffer (pH 3.2) containing 0.2 mM sodium octyl sulfate, 0.1 mM EDTA, and 15% v/v methanol (Mallinckrodt, Mexico), filtered (0.45 mm pores) and degassed prior to its use. The flow was set at 1.2 ml/min and the determinations were made at room temperature. An electrochemical detector (Metrohm 653) was used at a sensitivity of 10 nA, full scale. The monoamines (DOPAC, NE, 5-HIAA, DA, and 5-HT) were oxidized with a glassy carbon electrode at a potential of 850 mV relative to the Ag/AgCl reference electrode. The peaks generated by the compounds were recorded and integrated with a Hewlett Packard 3396 integrator. External standards (Sigma Chem. Co.) were used to construct a calibration curve for each of the monoamines. Brain Regional Arsenic and Lead Analysis Fresh tissue samples were weighed and solubilized with a mixture of 2 ml of nitric acid and 0.5 ml of perchloric acid and heated during 6 h at 70C. After evaporation, the residue was resuspended in nitric acid 0.2% with ammonium phosphate 0.5% for lead analysis, while for arsenic content determination 3% hydrochloride acid was used. Quantification was carried out using a Perkin-Elmer atomic absorption spectrophotometer (model 2380, Norwalk, CT) by the graphite furnace absorption technique (15,55) for lead, and by the hydride evolution-atomic absorption technique for arsenic (19,20,21,32, 53). As quality control, KODAK TEG-50B and NBS-SRM 1577 bovine liver standards were analyzed in parallel with the brain samples. Recoveries for KODAK standards were 90 11% and 94.8 5.6% (mean SD, n 8) for lead and arsenic, respectively. For NBS-SRM standards, recoveries were 96.8 10.5% for lead and 93.4 6.7% for arsenic (mean SD, n 8). Results were expressed in ng/mg of tissue, wet weight. Statistical Analysis Data were subjected to statistical analysis by means of the SPSS/PC program. Exploratory analysis of the data indicated a parametrical test to compare the experimental groups. Sets of data without normal distribution were log transformed. The data for each region were subjected to one-way analysis of variance. Groups of data showing a significant effect ( p 0.05) of the treatments were further analyzed by means of a multiple comparison procedure (Tukeys HSD test). The statistical comparison of regional metals content were performed by means of Students t-test. Differences among groups were considered significant at p-values under 0.05.
RESULTS

Animals and Metal Administration Male BALB/c mice, bred in-house, weighing 2530 g, were maintained on a standard 12-h lightdark cycle with food and water ad lib. Animals were housed in groups of 6 to 10. Two low oral arsenic doses, 11 and 13.8 mg/kg were employed in accordance with reports in the literature demonstrating vascular, immunological, renal, or neurological effects in rodents after subchronic arsenic oral exposure to this range of concentrations (3,6,30,37). The selection of doses was based on an acute lethal dose (LD50) study. Single intraperitoneal doses ranging from 11 to 18.8 mg/kg (sodium arsenite) were administered to five groups of six animals each. Then, the doses that were within the 95% confidence interval for LD0 (11 mg/kg) and LD10 (13.8 mg/kg) were chosen to study arsenic and arseniclead effects upon central monoaminergic systems. A lead dose of 116.4 mg/kg/day of lead acetate was selected to perform this study, in accordance with previous reports in the literature of behavioral and neurochemical effects on rodents (23,35). This dose was chosen to achieve the blood lead concentrations at which effects upon the central nervous system are observed in adult humans (30), and it corresponds to a LD10 for single intraperitoneal administration. Also, it was important not to reach blood lead levels beyond 100 mg/dl, to avoid an effect of lead on body weight (7). The mixture group received arsenic (13.8 mg/kg) and lead (116.4 mg/kg) together. The control group received only the vehicle (distilled water). These doses were administered through gastric intubation during 14 consecutive days to groups of 1620 animals. One hour after the last dose, animals were killed by cervical dislocation. Half of the group was destined for determination of the regional metal content, while monoamine quantification was carried out in the other half. In parallel, a group of 12 animals receiving the same lead treatment (116 mg/kg/day) was destined for estimation of blood lead levels. Analysis of Monoamines and Their Metabolites Discrete brain areas (hypothalamus, medulla and pons, midbrain, striatum, hippocampus, and cortex) were dissected out according to the procedure described by Glowinski and Iversen (24). Dissected tissues were weighed and immediately

First, we explored the effects of two low doses of arsenic (11 and 13.8 mg/kg/day). Both intoxicated groups showed detectable levels (lowest detectable level was 0.2 ng/mg of tissue) of arsenic in all regions tested, whereas the control group did not (Table 1). Arsenic concentration in the hypothalamus was between two and three times higher than in the rest of the

LEAD AND ARSENIC EFFECTS ON CENTRAL MONOAMINES TABLE 1

REGIONAL ARSENIC AND LEAD CONCENTRATIONS Region As (11 mg/kg) As (13.8 mg/kg) As (Mixture Group) Pb (116.4 mg/kg) Pb (Mixture Group)

491

Hypothalamus Medulla and pons Midbrain Striatum Hippocampus Cortex

1.21 .18 0.39 .03 0.34 .01 0.43 .04 0.51 .06 0.37 .03

0.98 .11 0.45 .08 0.32 .07 0.67 .14 0.35 .06 0.52 .07

0.61 .04* 0.35 .04 0.31 .03* 0.40 .05* 0.33 .05 0.27 .02

7.03 1.78 2.61 0.96 2.39 0.34 2.35 0.52 2.16 0.41 3.97 0.75

10.67 3.46 5.63 0.93 5.53 1.17 3.75 0.86 5.72 1.26 5.68 1.58

Values are means SEM (n 610) in nanograms per milligram of tissue (wet weight). No significant differences were found between the two arsenic doses. * p 0.05 p 0.01 p 0.001, by two-tailed Students t test.

regions. The components of the brain stem: pons, medulla, and midbrain, showed a very similar concentration. Somewhat lower amounts were found in the cortex and striatum, while the hippocampus showed the lowest concentration. There were no significant differences in regional arsenic content between the two groups. However, only one significant alteration in monoamine content was found at the lowest dose tested (Table 2). The observed changes consisted mainly in regional increases of DA, 5-HT, or their metabolites and a decrease of NE. Although the striatum was the only region showing a significant enhancement of DA concentration (72 11.5%, p 0.013), the hypothalamus and cortex showed important increases in DOPAC concentration, in the range of 150 to 230%. In the cortex, only the lower dose provoked a significant

change ( p 0.023). 5-HT showed a dual change at the medulla and pons. Because 5-HT levels showed a tendency to decrease at the lower dose (38 10%), the effect of the higher dose was a significant increase of 5-HT concentration compared to the lower dose (52 21%, p 0.002). A significant enhancement of the metabolite 5-HIAA was observed in the midbrain and striatum (91 31%, p 0.024 and 150 25%, p 0.005, respectively). By contrast, NE was significantly reduced in the hypothalamus (44 9%, p 0.01). Although the blood level of lead reached 79.3 4.9 g/dl (mean SEM, n 12) the animals showed neither signs of intoxication nor body weight loss. We did not find detectable levels (lowest detectable level was 1.0 ng/mg of tissue) of lead in any brain region of the control group, while after exposure

TABLE 2
BRAIN REGIONAL MONOAMINE CONCENTRATIONS AFTER ARSENIC TREATMENTS Hypothalamus Medulla and pons Midbrain Striatum Hippocampus Cortex

DA Control As (11 mg/kg) As (13.8 mg/kg) DOPAC Control As (11 mg/kg) As (13.8 mg/kg) 5-HT Control As (11 mg/kg) As (13.8 mg/kg) 5-HIAA Control As (11 mg/kg) As (13.8 mg/kg) NE Control As (11 mg/kg) As (13.8 mg/kg)

0.72 0.11 0.50 0.10 0.48 0.07 0.06 0.01 0.15 0.05 0.20 0.02* 1.42 0.23 1.51 0.41 1.46 0.18 0.47 0.02 0.34 0.07 0.46 0.03 3.70 0.44 2.60 0.25 2.07 0.33* 0.79 0.08 0.50 0.08 1.20 0.17 0.41 0.05 0.35 0.07 0.63 0.11 1.03 0.17 0.77 0.14 0.54 0.07

0.33 0.11 0.32 0.08 0.51 0.17 0.09 0.01 0.16 0.02 0.18 0.02 0.99 0.11 0.95 0.27 1.69 0.30 0.45 0.06 0.55 0.07 0.86 0.14* 0.76 0.10 0.66 0.18 0.83 0.16

5.24 0.98 7.23 0.99 9.05 0.63* 0.37 0.06 0.25 0.07 0.53 0.03 0.54 0.14 0.76 0.21 0.97 0.14 0.16 0.05 0.24 0.05 0.40 0.04* 0.73 0.19 0.49 0.13 0.67 0.18 0.54 0.08 0.37 0.07 0.62 0.10 0.26 0.05 0.19 0.03 0.33 0.07 0.61 0.13 0.29 0.06 0.39 0.13

0.79 0.11 0.68 0.22 0.66 0.22 0.06 0.01 0.15 0.04* 0.11 0.01 0.44 0.05 0.43 0.09 0.58 0.09 0.08 0.02 0.12 0.02 0.13 0.02 0.40 0.09 0.23 0.07 0.39 0.11

Effect of arsenic treatment on monoamines of several brain regions. Values are means SEM (n 610) in micrograms per gram of tissue (wet weight). *Significant differences ( p 0.05, Tukeys test) vs. control group. Significant difference between doses ( p 0.05 vs. As (11 mg/kg), Tukeys test).

492 to toxicants there were detectable lead levels in all regions tested. The administration of 116.4 mg/kg leadacetate resulted in a two- to threefold increase in lead accumulation in the hypothalamus compared to the other regions. The pons, medulla, and striatum showed the lowest concentrations, while intermediate concentrations were found in the cortex and hippocampus (Table 1).

MEJA ET AL. Compared to the control group, the dose of lead employed provoked a significant increase only of DOPAC in the hypothalamus (0.06 0.01 to 0.22 0.02 g/g, p 0.001). Given that most of the significant alterations due to arsenic were observed at the highest dose employed (13.8 mg/kg/day), the mixture group received this dose combined with 116.4 mg/kg/ day. The first sign of interaction from the combined adminis-

FIG. 1. Influence of lead, arsenic, and leadarsenic mixture treatments on (A) DA and (B) DOPAC in several brain regions. Mice were treated with distilled water, arsenic (13.8 mg/kg), lead (116.4 mg/kg), and the arsenic-lead mixture (13.8 mg/kg and 116.4 mg/kg for arsenic and lead, respectively) Each bar represents the mean SEM of 6 to 10 mice. Hyp, hypothalamus; m&p, medulla and pons; mid, midbrain; str, striatum; hpm, hippocampus; ctx, cortex. (A)*p 0.05 vs. control group. (B)*p 0.05 vs. control group. In addition, for ctx: As group was significantly different from Pb and AsPb groups.

LEAD AND ARSENIC EFFECTS ON CENTRAL MONOAMINES tration of metals was the concentration of lead reached in the brain regions. The administration of 116.4 mg/kg of leadacetate together with 13.8 mg/kg arsenic resulted in a higher accumulation of lead in the brain, as shown in Table 1. The greater lead brain content of the mixture group was accompanied by a decreased arsenic accumulation. When compared with the ar-

493

senic group, the concentration of arsenic in the mixture group was 2540% lower in the hypothalamus, midbrain, and striatum, whereas in the cortex it was almost 50% lower. Arsenic combined with lead provoked significant changes in all three monoamines and their metabolites. These changes were very similar to those elicited by arsenic alone. An aug-

FIG. 2. Comparison of lead, arsenic, and leadarsenic mixture treatments on (A) 5-HT and (B) 5-HIAA content of several brain regions. Mice were treated with distilled water, arsenic (13.8 mg/kg), lead (116.4 mg/kg), and the arseniclead mixture (13.8 mg/kg and 116.4 mg/kg for arsenic and lead, respectively). Each bar represents the mean SEM of 6 to 10 mice. Hyp, hypothalamus; m&p, medulla and pons; mid, midbrain; str, striatum; hpm, hippocampus; ctx, cortex. (A) *p 0.05 vs. control group. In addition, for str: As group p 0.05 vs. Pb group. (B) *p 0.05 vs. control group.

494

MEJA ET AL.

FIG. 3. Effect of lead, arsenic, and lead arsenic mixture treatments on NE content of several brain regions. Mice were treated with distilled water, arsenic (13.8 mg/kg), lead (116.4 mg/kg), and the arseniclead mixture (13.8 mg/kg and 116.4 mg/kg for arsenic and lead, respectively). Each bar represents the mean SEM of 6 to 10 mice. Hyp, hypothalamus; m&p, medulla and pons; mid, midbrain; str, striatum; hpm, hippocampus; ctx, cortex. *p 0.05 vs. control group. For hpm, in addition: AsPb group was significantly different from Pb group.

mentation of serotonergic content and that of its metabolite was observed in several structures. Significant increases of 5-HT were observed in the cortex (from 0.44 0.05, control vs. 0.84 0.18 g/g, mixture group, p 0.045), and midbrain (from 0.99 0.11, control vs. 1.98 0.27 g/g, mixture group, p 0.03). Also, 5-HIAA showed significant augmentations in the striatum (from 0.16 0.05, control vs. 0.47 0.09 g/g, mixture group, p 0.008) and midbrain (from 0.45 0.06 control vs. 1.04 0.07 g/g, mixture group, p 0.002). In the case of DA, the change induced by the mixture was almost identical to that elicited by arsenic alone. DA augmented in the striatum up to 8.74 0.86 g/g, from a basal level of 5.24 0.98 g/g (p 0.039). The mixture elicited a DOPAC augmentation in the hypothalamus in a similar proportion to that of lead and arsenic alone (from 0.06 0.01 to 0.22 0.03 g/g, p 0.001). Finally, the mixture group showed a decrease of NE concentration in the hypothalamus (from 3.7 0.44 to 2.02 0.23 g/g, p 0.032), which was an effect similar to that elicited by arsenic alone (Figs. 1 and 2). In addition, NE diminished in the hippocampus (from 0.61 0.13 to 0.23 0.05 g/g, p 0.019). While a significant NE diminution was observed in the hypothalamus of the arsenic group (from 1.03 0.17, control to 0.54 0.07 g/g, arsenic group, p 0.037), NE level in the medulla and pons from the mixture group was not significantly different from the control group (Fig. 3).

Table 3 shows the number of monoamines or metabolites affected by each treatment in each brain region. While lead alone altered just one monoamine in the hypothalamus, the effects of the mixture were more widespread and more severe, affecting five out of the six studied regions. The alterations produced by the mixture were very similar to those elicited by arsenic alone; however, the combined administration of lead and arsenic provoked alterations of NE in hippocampus and 5-HT in midbrain and cortex. These modifications were not elicited by either metal alone.
DISCUSSION

In the first part of this study, we examined the effects of the exposure of adult mice to two low doses of arsenic. Results of the present study support a regional alteration of monoamine metabolism induced by low-level arsenic exposure and consisted of a decrease of NE and increases of DA, 5-HT, or their metabolites, mainly at the regions bearing the highest content of these substances. In the second part, we administered a single mixture of arsenic and lead, comparing the effects with those of the exposure to either metal alone. The first important finding was the increase in regional lead levels when the animals were simultaneously exposed to arsenic and lead (Table 1). The observed increase was accompanied by a significant decrease of arsenic levels in some regions. Con-

TABLE 3
SUMMARY OF THE MONOAMINES AND/OR METABOLITES AFFECTED BY THE DIFFERENT TREATMENTS IN EACH BRAIN REGION Hypothalamus Medulla and pons Midbrain Striatum Hippocampus Cortex

Arsenic Lead Arsenic-lead

NE DOPAC DOPAC NE DOPAC

NE

5-HIAA 5-HT 5-HIAA

DA 5-HIAA DA 5-HIAA NE

DOPAC 5-HT

Comparison of the significant alterations elicited by three subchronic (14 days) oral treatments: arsenic (31.8 mg/kg as sodium arsenite), lead (116.4 mg/kg of lead acetate), and arsenic-lead (13.8 mg/kg of arsenic plus 116.4 mg/kg of lead acetate). Signs indicate increase () or decrease () vs. control values (vehicle).

LEAD AND ARSENIC EFFECTS ON CENTRAL MONOAMINES cerning the regional changes in monoamines and their metabolites, while lead increased only DOPAC concentration in hypothalamus, the administration of the mixture provoked a total of eight changes comprising augmentations of DA, DOPAC, 5-HT, or 5-HIAA in the hypothalamus, midbrain, striatum, and a decrease of NE in hypothalamus and hippocampus. These changes were similar to those produced by arsenic alone; however, the mixture elicited alterations of NE in the hippocampus and 5-HT in cortex and mibrain that were not produced by either metal alone. The observed changes in lead and arsenic accumulation when both are administered together represent further evidence of the complex chemical interactions of mixtures. We have previously reported a similar interaction for another single mixture, arseniccadmium (16,61). Our results agree with previous studies in the sense that they indicate an influence of lead in some step of the metabolism of brain biogenic amines, especially at the hypothalamic level (40), and they suggest a susceptibility of the dopaminergic system to lead insult (28,38,41). However, the extensive literature on this subject includes reports of lead-induced changes in monoaminergic systems in almost every direction (increase, decrease, no change) (18,46,49,52,58), depending upon the length of the treatment and age of exposition, as well as on the animal species and doses employed (27,51). Concerning the arsenic effects, the comparison of our results with some previous reports on arsenic effects on brain biogenic amines revealed a few similarities, for instance, a tendency for 5-HT and 5-HIAA to increase, especially in the striatum, as well as a higher susceptibility of the hypothalamus to show alterations in response to arsenic treatment (33,42). The decrease of NE in the hypothalamus has also been reported, but not consistently (33,36). The rest of the monoamines measured varied in a different direction, certainly because of the differences in treatments and age of the animals, as well as in the arsenic compound employed. Itoh et al. (33) used arsenic trioxide (3 and 10 mg/kg, 14 days) and reported dose-dependent changes of the monoamines NE (increase in cortex), DA (increase in hippocampus), 5-HT (increase in hypothalamus, hippocampus, striatum, and cortex). Nagaraja and Desiraju (42) administered sodium arsenate (5 mg/kg, 60 days), reporting a diminution of DA and an increase of 5-HT in striatum while NE was reduced in hippocampus. Because sodium arsenate displays ionic similarity to phosphate, it is possible that the arsenate affected some processes that the arsenite does not influence (10). Given that in some regions the observed increases in DA and 5-HT were paralleled by increases in their respective metabolites, DOPAC and 5-HIAA, it is possible that both the synthesis (tyrosine hidroxylase activity) and perhaps also metabolism (MAO activity) were augmented. Toxic metal-induced increases of MAO activity have been reported for thallium (45) and manganese (43), and this could be the case for arsenic and

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lead as well. The selective increase of 5-HT due to the mixture exposure is an important finding because the modulation of cortical activity, of which 5-HT and acetylcholine are the main responsible agents, is the basis of arousal and cognitive functions, especially in terms of learning and memory (8,34,56). Future experiments will address a possible cortical serotonergic dysfunction that may partially account for the behavioral and sleep disturbances observed in children (17), because studies reported by others showed even more important serotonergic effects when developing mice received a similar treatment (33). The observed regional increase in neurotransmitter content is difficult to extrapolate to changes in synaptic function. Lead has been reported to produce alterations in intracellular calcium regulation (26), and at least for DA, 5-HT and acetylcholine lead induced an increase of spontaneous release from striatal or cortical synaptosomes (5,41,54). Also, cadmium is able to increase DA release (31). These results suggest that lead is not acting specifically at the level of a given kind of neuron. Because most of the modifications of regional transmitter content observed in this study took place in the region where a given transmitter is more abundant, it is possible that a generalized alteration of several types of neurons became observable only in the regions where the proportion of affected cells was highest. Although most of the alterations observed in this study were augmentations of DA, 5-HT, and/or their metabolite levels, NE concentration showed a diminution in response to arsenic and to the mixture. As a working hypothesis to explain NE decreases, we suspect damage at the hypothalamic level, possibly strong enough to alter the neuroendocrinal signalling between this structure and the locus coeruleus. The hypothalamus is the main source of input to the noradrenergic structures of the brain stem (11,39), and changes of several pituitary hormones in response to toxic metals exposure have been reported elsewere (29,47). Modifications in the neuroendocrine signalling are a possibility that is interesting to explore further. In conclusion, we demonstrated an interaction of arsenic and lead on the monaminergic systems of the adult mouse. This study supports the view that few results from singleexposure testing of a toxic substance can be extrapolated to the real effects of that toxicant in the presence of other substances (59,60). Therefore, to asses the neurotoxic alterations produced by a chemical, further research will need to be conducted using chemical mixtures.
ACKNOWLEDGEMENTS

This work was supported by grants 2168-M9303, F577-A9402, and a repatriation fellowship to M.E.J.C. from the Consejo Nacional de Ciencia y Tecnologa (CONACYT). The authors acknowledge the technical assistance of Laura Osorio Rico and Juan Manuel Delgado C.

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