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Pb and Cd poisoning during development alters cerebellar and striatal function in rats
Toxicology 176 (2002) 59 – 66 www.elsevier.com/locate/toxicol
Pb and Cd poisoning during development alters cerebellar and striatal function in rats M. Teresa Antonio, Noelia Lo´pez, M. Luisa Leret * Department of Animal Biology II, Faculty of Biology, Complutense Uni6ersity, 20840 Madrid, Spain Received 1 November 2001; received in revised form 28 March 2002; accepted 28 March 2002
Abstract The present study was designed to examine more fully the neurochemical and behavioral interactions that derive from continued lead and cadmium poisoning in pups, whose mothers were exposed via drinking water (300 mg/l of Pb and 10 mg/l of Cd) throughout pregnancy and lactation. At weaning, these metals produced an increase in DOPAC, 5-HT and 5-HIAA contents in cerebellum, but the monoamine contents in striatum remained unaltered. The cerebral energetic metabolism was modified by the Cd– Pb exposition only in striatum. On the other hand, the Na+/K+-ATPase activity was inhibited significantly in both regions at PN21, whereas the alkaline phosphatase activity was not affected by the treatment. The intoxicated animals showed a short-term normocitic anemia, but revealed long-term alterations in the motor activity in open-field, where they showed an increase in both ambulating and rearing. So, it can be concluded that perinatal exposure to lead and cadmium provoke neurochemical alterations in cerebellum and striatum that can be related with the changes in motor activity observed in the adulthood. © 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Lead; Cadmium; Cerebellum; Striatum; ATP; Monoamines; Motor activity
1. Introduction Exposure to lead (Pb) and cadmium (Cd) starts in the fetal life and are particularly dangerous for the central nervous system in long-term, low-dose exposure in mammals, especially during the early development. Both metals cross the placenta, but the transplacental transport of cadmium is limited, probably due to the binding to metaloth* Corresponding author. Tel.: +91-39-44-896; fax: + 9139-44-935. E-mail address: [email protected] (M.L. Leret).
ionein (MT), a protein rich in sulphydril groups implicated in zinc and copper homeostasis. During lactation, both metals are excreted to maternal milk, and this is the period of the greatest bioavailability of these metals because the intestinal absorption is higher in pups than in adults (Bhattacharyya, 1983). Previous studies have demonstrated that Pb and Cd can interact with gastrointestinal absorption of calcium, iron and zinc. This interaction with divalent cations is considered one of the molecular bases for the toxicity of these heavy metals. The replacement of Zn in numerous en-
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zymes and the decrease in the availability of iron could be the mechanism by which Pb and Cd exert their toxic effects, as is the case of the anemia associated with their poisoning (Goyer, 1995). On the other hand, calcium is an essential nutrient, which is required in multiple cellular and physiological functions, such as cell adhesion, signal transduction, neurotransmission, etc. Lead and cadmium can substitute it in calmodulin and alter its correct function, inhibit the Ca2 + bombs and channels, and replace it in various calcium receptors. The interaction with Ca2 + in the neurotransmission process, or the impairment in its homeostasis, have been often implicated in the alterations described in the content and release of neurotransmitters (Vig and Nath, 1991). Both metals have high affinity for the free sulphydril groups in enzymes and proteins and its binding can alter their correct function in numerous and not related process. Besides, the binding of Pb and Cd to the SH groups of glutation could be implicated in the induction of oxidative stress associated with Pb and Cd poisoning (Bagchi et al., 1997). All the proposed mechanisms by which these metals specially affects behavior, however, continue to engender controversy. When the exposition occurs in early life, the behavioral effects of Pb and Cd usually persist even in adult life. The alteration in the motor activity is one of the most studied effects of the intoxication, but learning impairments, in particular caused by lead, has been described at very low doses and included a decrease in the intelligence quotient as well as in attention capacity and memory (Cory-Sletcha, 1995). The aim of this work is the study of the effects that the combined exposure to lead and cadmium causes in rats after a long-term exposition during gestation and lactation, the critical period in the development of central nervous system. Since the poisoning with these metals has been associated with motor behavior alterations, attention was given to the neurochemical study of cerebellum and striatum, two important regions implicated in the control of this aspect of behavior.
2. Methods Albino rats from a laboratory-inbreed Wistar strain (200–250 g) were used throughout the experiments. Females in estrous were mated with males and the pregnancy was confirmed by the presence of sperm in vaginal smears. The animals were maintained under standard laboratory conditions (12-h light:12-h dark, 219 1 °C) and under the supervision of a licensed veterinarian in accordance with the principles set forth in the NIH Guide for the Care and Use of Laboratory Animals. One group of females (Pb– Cd group) was exposed to 300 mg/l of lead and 10 mg/l of cadmium, as acetate, in the drinking water, while the control group was maintained with distilled water ad libitum, from the beginning of pregnancy until weaning (postnatal day 21: PN21). At parturition, litters were culled to 10–12 pups. Maternal weight, fluid and food intake were monitored throughout all the treatment period and neonate weight gain assessed in the postnatal period. The pups were sacrificed by decapitation at weaning. Blood samples were obtained immediately, brain excised and the cerebellum and striatum quickly dissected as previously described (Molina-Holgado et al., 1993). Monoamine levels were estimated in both cerebellum and striatum at PN21. For this purpose, samples were homogenized in cold 0.2 N perchloric acid containing 0.4 mM sodium bisulphite and 0.4 mM EDTA. 3,4Dihydroxybenzylamine was also added to each sample as internal standard. The homogenates were centrifuged (13 000 rpm, 5 min at 4 °C) and supernatants were used for determination of levels of dopamine (DA), 3,4-dihydroxiphenilacetic acid (DOPAC), 5-hydroxytryptamine (5-HT) and 5hydroxyindolacetic acid (5-HIAA) using high performance liquid chromatography with electrochemical detection (HPLC-ED) (Column Spherisorb ODS2-22 cm× 4.6 mm after precolumn 10 glc 4 ODS2). The mobile phase for the elution consisted of 0.05 M monopotassium phosphate, 0.1 mM EDTA, 1 mM heptane sulphonate and 8% methanol, with a pH adjusted to 3.2 with acetic acid. Standards were run concurrently and concentrations of unknowns were determined by
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comparison to peak areas of standards after correction for recovery of the internal standard (Leret et al., 1993). Nucleotide level determination was done in cerebellum and striatum at PN21.The samples were homogenized in cold perchloric acid 6% containing 0.8 mM EDTA, centrifuged (10 000 rpm, 10 min, 4 °C), and supernatants adjusted at pH 4.6 with KOH 6 M for the determination by HPLC with UV detection (Column Bondclone 10C18-300×3.9 mm). The mobile phase consisting of 0.2 M monopotassium phosphate adjusted to pH 6.0 with NH4OH. (Rotlla´ n et al., 1986). The total adenine nucleotides (TAN) was obtained as the sum of ATP, ADP and AMP concentrations, and the adenylate energy charge (AEC) using the formula: (ATP +1/2ADP)/TAN. Some samples were used for the estimation of ATPase activity. Cerebellum and striatum were homogenized in Tris– HCl 40 mM pH 7.4. For the total ATPase activity, the reaction was made by addition of 0.1 ml of this homogenate in 0.8 ml of medium consisting of: MgCl2 6 mM, EDTA 1 mM, NaCl 100 mM and KCl 20 mM in Tris – HCl 40 mM pH 7.4. The same medium, after addition of 1 mM ouabain, was used for the determination of Mg2 + -ATPase. The reaction was initiated by addition of 5 mM ATP, and it was stopped with cold TCA 10%. Measuring the liberated phosphorus (Pi) by colorimetric assay, the enzymatic activity was estimated. (Zaheer et al., 1968). Alkaline phosphatase activity in cerebral tissues was measured also in homogenates of cerebellum and striatum following the p-nitrophenol method (Kuftinec and Miller, 1972). Protein levels were determined (Lowry et al., 1951) for the calculation of the specific activity of the studied enzymes. Some animals, selected randomly, were maintained with distilled water ad libitum until 75 days of age, when were submitted to behavioral testing. Open-field testing was performed during a 5 min test. Exploratory activity (ambulations as number of line crossings) and rearing were noted. Behavioral results were compared by means of Mann– Whitney test after Kruskal– Wallis one-way analysis of variance (ANOVA). The results are expressed as means9standard deviation (S.D.) or means9 standard error or the
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mean (S.E.M.) (in the figures). Statistical data were analyzed using Student’s t-test, with P B 0.05.
3. Results During the treatment period, the intoxicated mothers showed a daily gain of weight equivalent to the control group. Likewise, the volume drinking and the food consumption were equal in both groups during all gestation and lactation (data not shown). The ingested dose by mothers in gestation was of 31.649 5.39 mg Pb and 1.029 0.20 mg Cd/kg weight per day. During lactation, the administered daily dose was 64.989 16.01 mg Pb and 2.179 0.87 mg Cd/ kg weight per day. The intoxicated pups showed, since birth, a lineal gain of weight statistically equal to the control pups (Fig. 1). At weaning neither the absolute or relative brain weight were affected by the metals exposure (Table 1). The Hb content in the plasma of the intoxicated group decreased significantly (8.939 1.18 g/dl in control group and 6.739 1.34 g/dl in experimental group). The hematocrit (26.59 3.74% control and 22.19 0.74% Cd–Pb group), as well as the number of red blood cells (in millions/mm3: 4.919 1.37 in control group and
Fig. 1. Weight gain in pups from birthday until postnatal day 21. Results are expressed as mean 9S.E.M. (n =8 – 11).
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Table 1 Results obtained in absolute and relative brain weight (OSI defined as brain weight×1000/body weight) and alkaline phosphatase activity in striatum and cerebellum
Brain weigth (g) Brain OSI Alkaline phosphatase activity in striatum (mmol pnitrofenol/mg prot per min) Alkaline phosphatase activity in cerebellum (mmol pnitrofenol/mg prot per min)
Control
Pb+Cd
1.3390.07 40.15 9 3.75 3.4 9 0.36
1.34 90.04 38.75 9 3.20 3.09 0.4
3.32 90.16
3.08 90.08
higher (+ 158%) in exposed animals, with a significative increase of turnover DOPAC/DA. The serotoninergic system was more affected because there was a significative increase of both 5-HT (+ 82%) and 5-HIAA ( +86%) in the cerebellum, without changes in its turnover (Table 3). The results obtained in the open-field test are showed in Fig. 5. The exposed animals present in the adulthood an increase of total as well as external ambulation, while the rearing and internal ambulation were not affected by the metals exposition.
Results are expressed as mean 9S.D. (n =8–11).
3.2091.21 in experimental group) are also reduced significantly. In conclusion, the intoxicated pups show a short-term normocitic anemia because all these parameters return to normal values at postnatal day 75 (data not shown). Fig. 2 shows the results obtained in the ATPase activity estimated in the cerebellum and striatum of pups at PN21. In both regions Pb and Cd provoked a significative reduction of Na+/K+ dependent ATPase ( −33% in cerebellum; − 23% in striatum). This effect is present only in shortterm because the activity of this enzyme was equal to the control group in both regions at PN75. Neither the total nor the Mg2 + -dependent ATPase activities were affected. The activity of the other enzyme quantified in brain, the alkaline phosphatase, was not modified for Pb and Cd intoxication neither in any regions any both ages studied (Table 1). The cerebral energetic metabolism was modified by the exposition only in the striatum of pups (Fig. 3). The ATP as well as ADP and AMP content is reduced in this area, but only the decrease of ATP was significative. Thereby, neither the AEC nor the ATP/ADP ratio were affected, but the TAN was lower than in control group (Table 2). In contrast, the monoamine contents were modified by the metals exposure only in the cerebellum (Fig. 4). The DA content was equivalent in control and treated groups, but the DOPAC was
Fig. 2. Effects of Pb and Cd on the Na+/K+-ATPase activity in Striatum (A) and Cerebellum (B) at PN21. Results are expressed as mg Pi/mg protein per h (mean 9S.E.M.), *PB 0.05, **PB 0.01 (n =8 – 11).
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Fig. 3. Nucleotide levels of Cerebellum (A) and Striatum (B) of pups at PN21. Results are expressed as mean 9S.E.M., **PB 0.01 (n =8 – 11).
4. Discussion Although heavy metals ions are known to be toxic to the central nervous system, the immature
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Fig. 4. Levels of monoamines in Cerebellum (A) and Striatum (B) of control and intoxicated pups at PN21. Results are expressed as mean 9S.E.M., *PB 0.05 (n = 9 – 10).
blood–brain barrier and the absence of protein complexes that sequester metals in mature tissues result in increased vulnerability of the fetal brain
Table 2 Total adenine nucleotides (TAN), ATP/ADP ratio and Adenylate Energy Charge (AEC) Control
Pb+Cd
Cerebellum TAN ATP/ADP AEC
146.719 24.98 1.319 0.63 0.369 0.12
158.90 956.45 1.32 90.78 0.42 90.09
Striatum TAN ATP/ADP AEC
205.83 943.74 2.03 90.81 0.42 90.09
146.94 971.59* 1.32 9 0.70 0.33 90.14
*PB0.05, Student’s t-test, results are expressed as mean 9 S.D. (n = 7–8).
Table 3 DA and 5-HIAA/5-HT ratio Control
Pb+Cd
Cerebellum DOPAC/DA 5-HIAA/5-HT
2.16 91.01 1.14 9 0.36
6.54 93.30* 1.81 90.8
Striatum DOPAC/DA 5-HIAA/5-HT
0.22 90.04 0.95 90.38
0.26 9 0.12 1.04 9 0.38
Results are expressed as mean 9S.D. (n =6–9), *PB0.05, Student’s t-test.
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Fig. 5. Results obtained in open-field test conducted at PN75 in control and intoxicated animals. Results are expressed as mean9 S.E.M., *P B0.05 (n=9–10).
to lead and cadmium. In this sense, our results confirm that Pb and Cd may exert an inhibitory effect directly on Na+/K+-ATPase activity. This enzyme plays a vital role in linking the extracellular signals to the intracellular medium in neural tissues, so the inhibition of this enzyme by Cd and Pb could be damaging to the brain cells leading to an earlier stage of edema, followed by a later stage of degeneration and necrosis. Several previous works have shown that perinatal exposition to these metals, even at doses lower than here, can inhibit the synaptosomal Na+/K+-ATPase (Rafalowska et al., 1996). Pb and Cd could bind to free SH groups and inhibit reversibly the enzyme, so that mechanism could explain why isoforms with subunits a2 and a3, limited to nervous tissue and cardiac muscle, are more susceptible to the inactivation for their higher content of cysteins (Fox et al., 1991). On the other hand, the in vitro studies indicates that Cd is a more potent inhibitor than Pb, related to its high affinity for the free SH (Carfagna et al., 1996). In our laboratory, we found also in cortex and cerebellum a significant inhibition of this enzyme after perinatal exposure to isolated Pb, although the rate of inactivation was lower than in this work (Antonio and Leret, 2000), suggesting an additive effect, in agreement with the in vitro studies (Carfagna et al., 1996). Alkaline phosphatase has been recently implicated in neuritogenesis processes, even in early
development, and also in mielinization processes, which in rats occurs rapidly in the first 2 weeks after birth. Likewise, it could participate on energetic metabolism and on transduction signal pathways (Kern and Audesirk, 1995; Shinozaki and Pritzker, 1996). A similar exposition to Pb in our laboratory, provoked a fall on its cerebral activity at PN12 (Antonio and Leret, 2000). This enzyme is Zn-dependent, so depletion on cerebral Zn caused by Pb and Cd (Gupta et al., 1993) and/or their binding to SH groups, could explain its inhibition. In our study no effects have been detected at this level, which could be related to the age of the animals, because the inhibition cited above coincided with the intense mielinization period, or with a possible antagonic effects between both metals. With respect to brain nucleotide levels, a decrease was observed in this study only in the striatum of exposed pups. In previous works conducted in our laboratory we observed that using lead alone intoxication protocol, striatum was also more affected (Antonio and Leret, 2000). Pb has been proposed to inhibit the oxidative phosphorilation, although the exact target is still unclear. Some authors maintain that, since synaptosomal respiration results unaltered after Pb poisoning, ATP depletion could be related to a structural and functional damage of mitochondrial membranes (Rafalowska et al., 1996), or to a delayed maturation of oxidative metabolism (Bull, 1983). Cadmium toxicity implicates the inhibition of the respiratory chain, both on I and II complexes (Miccadei and Floridi, 1993). Alteration of cerebral monoamines content has been frequently related to these heavy metals poisoning. Although the effects provoked by Cd are not fully understood, doses similar to ours are associated with an increase of monoamine levels (Ma´ rquez et al., 1999). Pb seems to have opposite effects, causing a drop in similar expositions. This antagonic effect has been contrasted in our laboratory by individual administration of the same doses of these metals (Antonio et al., 1996, 1998). Alteration in neurotransmitters release mechanisms, or in their synthesis and catabolism, seems to participate on these effects. Cd is able to stimulate cathecolamine synthesis, by activating
M.T. Antonio et al. / Toxicology 176 (2002) 59–66
the calmodulin dependent pathway, and even inhibiting catabolic enzymes, such as monoaminoxidases (Leung et al., 1992). In contrast, Pb could interact with some signal pathways, which are implicated in monoamine synthesis, as well as with tyrosine hydroxylase activity or the tetrahydrobiopterin synthesis (Ramin and Porter, 1997). Cotreatment with cadmium and lead difficult the interpretation of the results. The increase of indolamines and DOPAC in the cerebellum of intoxicated pups does not belong with the results obtained from perinatal administration of Pb at postnatal days 0, 5 and 12, where a decrease on monoamines content were observed (Antonio et al., 1996; Antonio and Leret, 2000). Our results are more comparable to those produced by Cd alone (Antonio et al., 1998). Two stages could be distinguished during the exposition period, according to the bioavailability of these metals. Throughout pregnancy, the availability of lead is higher, while during lactation takes place the highest capture and storage of Cd by the pups. Perhaps we are detecting the combined effects, although, due to their recent exposition, those related to Cd are more evident in the brain of the pups. Excepting the change in ATP levels, the cerebellum has been more sensitive to intoxication. It has been described the homogeneity of these metals in various regions of the brain (Widzowski and Cory-Sletcha, 1994)), so its higher sensitivity could be due to functional features. Thus, differences in both regions development must be considered, because this study was conducted in this critic period. Most of the cells appear in striatum during fetal life, when Cd exposure is lower, while the cerebellum development extends until advanced lactation, when both metals are fully available, and its higher susceptibility could be due to the combined exposition to lead and cadmium. The effects of lead and cadmium on neurobehavioral development have been extensively investigated in humans. Locomotion and conditioned responses dysfunctions, as well as decreased intelligence, hyperactivity, reduced learning/memory capacities and deficient IQ scores are the main symptoms observed in both children and adults exposed to lead and/or cadmium (Taylor and
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Ennever, 1993; Tong, 1998). In animals, behavioral effects of Cd are very controversial, but are normally associated with a decrease in ambulation and rearing in open-field test (De´ si et al., 1998). However, our results are similar to the effects described after lead poisoning (Ma, et al., 1999), and the interaction with dopaminergic system has been often involved in this hyperactivity (CorySletcha, 1995; Ma, et al., 1999). Morphological and electrophysiological alterations in cerebellum could be also underlying, such as molecular layer atrophy and decrease in granulate cells density and spontaneous discharge of Purkinje cells (Bo¨ rjklund et al., 1983). On the other hand, Pb could alter synaptic plasticity by interaction with adhesion molecules during early development (Murphy and Regan, 1999), which could explain the persistence of effects even in adult life. In conclusion, we have detected after perinatal exposure to low to moderate doses of Pb and Cd, neurochemical alterations in cerebellum and striatum at weaning, such as the decrease in enzymes activities and energetic metabolism, or the alteration in the monoamines content. An important finding in this study is that these effects could be responsible of the alterations in motor behavior observed in the adulthood of intoxicated rats.
References Antonio, M.T., Leret, M.L., 2000. Study of the neurochemical alterations produced in discrete brain areas by perinatal low-level lead exposure. Life Sci. 67, 635 – 642. Antonio, M.T., Martı´nez, S., Leret, M.L., Corpas, I., 1996. Neurotoxic effects of gestational administration of lowdose lead acetate. J. Appl. Toxicol. 16, 431 – 436. Antonio, M.T., Benito, M.T., Leret, M.L., Corpas, I., 1998. Gestational administration of cadmium alters the neurotransmitter levels in newborn rat brains. J. Appl. Toxicol. 18, 83 – 88. Bagchi, D., Vuchetich, P.J., Bagchi, M., Hassoun, E.A., Tran, M.X., Tang, L., Stohs, S.J., 1997. Induction of oxidative stress by chronic administration of sodium dichromate (chromium VI) and cadmium chloride (cadmium II) to rats. Free Radic. Biol. Med. 22, 471 – 478. Bhattacharyya, M.H., 1983. Bioavailability of orally administered cadmium and lead to mother, fetus and neonate during pregnancy and lactation: an overview. Sci. Total Environ. 28, 327 – 342.
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Bo¨ rjklund, H., Palmer, M.R., Lind, B., Hoffer, B.J., Olson, L., 1983. Postnatal lead exposure alters spontaneous cerebellar purkinje neuron discharge. Environ. Res. 31, 448 – 459. Bull, R.J., 1983. Delayed metabolic maturation of the cerebral cortex of rat pups derived from lead-treated dams. J. Toxicol. Environ. Health 11, 211 – 225. Carfagna, M.A., Ponsler, G.D., Muhoberac, B.B., 1996. Inhibition of ATP-ase activity in rat synaptic plasma membranes by simultaneous exposure to metals. Chem.-Biol. Interact. 100, 53 – 65. Cory-Sletcha, D.A., 1995. Relationships between lead-induced learning impairments and changes in dopaminergic, cholinergic, and glutamaergic neurotransmitter system functions. Annu. Rev. Pharmacol. Toxicol. 35, 391 –415. De´ si, I., Nagymajtenyi, L., Schultz, H., 1998. Behavioral and neurotoxicological changes caused by cadmium treatment of rats during development. J. Appl. Toxicol. 18, 63 –70. Fox, D.A., Rubinstein, S.D., Hsu, P., 1991. Developmental lead exposure inhibits adult rat retinal, but not kidney, Na+ /K + -ATPase. Toxicol. Appl. Pharmacol. 109, 482 – 493. Goyer, R.A., 1995. Nutrition and metal toxicity. Am. J. Clin. Nutr. 61S, 646 – 650. Gupta, A., Murphy, R.C., Chandra, S.V., 1993. Neurochemical changes in developing rat brain after pre-and postnatal cadmium exposure. Bull. Environ. Contam. Toxicol. 51, 12–17. Kern, M., Audesirk, G., 1995. Inorganic lead may inhibit neurite development in cultured rat hippocampal neurons through hyperphosphorilation. Toxicol. Appl. Pharmacol. 134, 111 – 123. Kuftinec, M.M., Miller, S.A., 1972. Alkaline and acid phosphatase activities during growth of long bones and mandibles. Calcif. Tissue Res. 9, 173 –178. Leret, M.L., Gonza´ lez, M.I., Arahuetes, R.M., 1993. Effects of maternal adrenal deprivation on the content on the content of catecholamines in fetal brain. Life Sci. 52, 1609 – 1615. Leung, T.K.C., Lim, L., Lai, J.C.K., 1992. Differential effects of metal ions on type A and B monoamine oxidase activities in rat brain and liver mitochondria. Metab. Brain Dis. 7, 139 – 146. Lowry, O.H., Rosenbrough, N.J., Farr, N.J., Randall, R.I., 1951. Protein measurements with the folin –phenol reagent. J. Biol. Chem. 193, 175 –265. Ma, T., Chen, H., Ho, I.K., 1999. Effects of chronic lead (Pb) exposure on neurobehavioral function and dopaminergic neurotransmitter receptors in rats. Toxicol. Lett. 105, 111 – 121.
Ma´ rquez, N., Esquifino, A.I., Canals, S., Lafuente, A., 1999. El cadmio es un potente disruptor neuroendocrino I. Alteraciones en el eje hipotala´ mico-hipofisario-testicular. Revista de Toxicologı´a 16, 148 – 149. Miccadei, S., Floridi, A., 1993. Sites of inhibition of mitochondrial electron transport by cadmium. Chem-Biol. Interact. 89, 159 – 167. Molina-Holgado, F., Molina-Holgado, E., Leret, M.L., Gonzalez, M.I., Reader, F.A., 1993. Distribution of indolamines and [3H]-paroxetine binding in rat brain regions following acute of perinatal 9D-Tetrahydrocannabinol treatment. J. Neurochem. Res. 18, 1183 – 1191. Murphy, K.J., Regan, C.M., 1999. Low-level lead exposure in the early postnatal period results in persisting neuroplastic deficits associated with memory consolidation. J. Neurochem. 72, 2099 – 2104. Rafalowska, U., Struzynska, L., Dabrowska-Bouta, B., Lenkiewicz, A., 1996. Is lead toxicosis a reflection of altered energy metabolism in brain synaptosomes? Acta Neurobiol. Exp. 56, 611 – 617. Ramin, S.M., Porter, J.C., 1997. A study of the cellular mechanism by which lead affects catecholamine secretion. Life Sci. 61, 1313 – 1321. Rotlla´ n, P., Liras, A., Llorente, P., 1986. A set of procedures for resolving purine compounds by reversed-phase high performance liquid chromatography: application to the study of purine nucleotide and nucleic acid metabolism. Anal. Biochem. 159, 377 – 385. Shinozaki, T., Pritzker, K.P.H., 1996. Regulation of alkaline phosphatase: implications for calcium pyrophosphate dehydrate crystal dissolution and other alkaline phosphatase functions. J. Rheumatol. 23, 677 – 683. Taylor, J., Ennever, F.K., 1993. Toxicological profile for cadmium, Atlanta, Agency for Toxic Substances and Disease Registry, US Public Health Service. Tong, S., 1998. Lead exposure and congnitive development: persistence and dynamic pattern. J. Paediatr. Child Health 34, 114 – 118. Vig, P.J.S., Nath, R., 1991. Interaction of metals with brain calmodulin purified from normal and cadmium exposed rats. Drug Chem. Toxicol. 14, 207 – 218. Widzowski, D.V., Cory-Sletcha, D.A., 1994. Homogeneity of regional brain lead concentrations. Neurotoxicology 15, 295 – 308. Zaheer, N., Iqbal, Z., Talwar, G.P., 1968. Metabolic parameters of ontogenesis of electrical activity in the brain. Sodium – potassium activated adenosine triphosphatase in developing chick embryo. J. Neurochem. 15, 1217 – 1224.
Pb and Cd poisoning during development alters cerebellar and striatal function in rats M. Teresa Antonio, Noelia Lo´pez, M. Luisa Leret * Department of Animal Biology II, Faculty of Biology, Complutense Uni6ersity, 20840 Madrid, Spain Received 1 November 2001; received in revised form 28 March 2002; accepted 28 March 2002
Abstract The present study was designed to examine more fully the neurochemical and behavioral interactions that derive from continued lead and cadmium poisoning in pups, whose mothers were exposed via drinking water (300 mg/l of Pb and 10 mg/l of Cd) throughout pregnancy and lactation. At weaning, these metals produced an increase in DOPAC, 5-HT and 5-HIAA contents in cerebellum, but the monoamine contents in striatum remained unaltered. The cerebral energetic metabolism was modified by the Cd– Pb exposition only in striatum. On the other hand, the Na+/K+-ATPase activity was inhibited significantly in both regions at PN21, whereas the alkaline phosphatase activity was not affected by the treatment. The intoxicated animals showed a short-term normocitic anemia, but revealed long-term alterations in the motor activity in open-field, where they showed an increase in both ambulating and rearing. So, it can be concluded that perinatal exposure to lead and cadmium provoke neurochemical alterations in cerebellum and striatum that can be related with the changes in motor activity observed in the adulthood. © 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Lead; Cadmium; Cerebellum; Striatum; ATP; Monoamines; Motor activity
1. Introduction Exposure to lead (Pb) and cadmium (Cd) starts in the fetal life and are particularly dangerous for the central nervous system in long-term, low-dose exposure in mammals, especially during the early development. Both metals cross the placenta, but the transplacental transport of cadmium is limited, probably due to the binding to metaloth* Corresponding author. Tel.: +91-39-44-896; fax: + 9139-44-935. E-mail address: [email protected] (M.L. Leret).
ionein (MT), a protein rich in sulphydril groups implicated in zinc and copper homeostasis. During lactation, both metals are excreted to maternal milk, and this is the period of the greatest bioavailability of these metals because the intestinal absorption is higher in pups than in adults (Bhattacharyya, 1983). Previous studies have demonstrated that Pb and Cd can interact with gastrointestinal absorption of calcium, iron and zinc. This interaction with divalent cations is considered one of the molecular bases for the toxicity of these heavy metals. The replacement of Zn in numerous en-
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zymes and the decrease in the availability of iron could be the mechanism by which Pb and Cd exert their toxic effects, as is the case of the anemia associated with their poisoning (Goyer, 1995). On the other hand, calcium is an essential nutrient, which is required in multiple cellular and physiological functions, such as cell adhesion, signal transduction, neurotransmission, etc. Lead and cadmium can substitute it in calmodulin and alter its correct function, inhibit the Ca2 + bombs and channels, and replace it in various calcium receptors. The interaction with Ca2 + in the neurotransmission process, or the impairment in its homeostasis, have been often implicated in the alterations described in the content and release of neurotransmitters (Vig and Nath, 1991). Both metals have high affinity for the free sulphydril groups in enzymes and proteins and its binding can alter their correct function in numerous and not related process. Besides, the binding of Pb and Cd to the SH groups of glutation could be implicated in the induction of oxidative stress associated with Pb and Cd poisoning (Bagchi et al., 1997). All the proposed mechanisms by which these metals specially affects behavior, however, continue to engender controversy. When the exposition occurs in early life, the behavioral effects of Pb and Cd usually persist even in adult life. The alteration in the motor activity is one of the most studied effects of the intoxication, but learning impairments, in particular caused by lead, has been described at very low doses and included a decrease in the intelligence quotient as well as in attention capacity and memory (Cory-Sletcha, 1995). The aim of this work is the study of the effects that the combined exposure to lead and cadmium causes in rats after a long-term exposition during gestation and lactation, the critical period in the development of central nervous system. Since the poisoning with these metals has been associated with motor behavior alterations, attention was given to the neurochemical study of cerebellum and striatum, two important regions implicated in the control of this aspect of behavior.
2. Methods Albino rats from a laboratory-inbreed Wistar strain (200–250 g) were used throughout the experiments. Females in estrous were mated with males and the pregnancy was confirmed by the presence of sperm in vaginal smears. The animals were maintained under standard laboratory conditions (12-h light:12-h dark, 219 1 °C) and under the supervision of a licensed veterinarian in accordance with the principles set forth in the NIH Guide for the Care and Use of Laboratory Animals. One group of females (Pb– Cd group) was exposed to 300 mg/l of lead and 10 mg/l of cadmium, as acetate, in the drinking water, while the control group was maintained with distilled water ad libitum, from the beginning of pregnancy until weaning (postnatal day 21: PN21). At parturition, litters were culled to 10–12 pups. Maternal weight, fluid and food intake were monitored throughout all the treatment period and neonate weight gain assessed in the postnatal period. The pups were sacrificed by decapitation at weaning. Blood samples were obtained immediately, brain excised and the cerebellum and striatum quickly dissected as previously described (Molina-Holgado et al., 1993). Monoamine levels were estimated in both cerebellum and striatum at PN21. For this purpose, samples were homogenized in cold 0.2 N perchloric acid containing 0.4 mM sodium bisulphite and 0.4 mM EDTA. 3,4Dihydroxybenzylamine was also added to each sample as internal standard. The homogenates were centrifuged (13 000 rpm, 5 min at 4 °C) and supernatants were used for determination of levels of dopamine (DA), 3,4-dihydroxiphenilacetic acid (DOPAC), 5-hydroxytryptamine (5-HT) and 5hydroxyindolacetic acid (5-HIAA) using high performance liquid chromatography with electrochemical detection (HPLC-ED) (Column Spherisorb ODS2-22 cm× 4.6 mm after precolumn 10 glc 4 ODS2). The mobile phase for the elution consisted of 0.05 M monopotassium phosphate, 0.1 mM EDTA, 1 mM heptane sulphonate and 8% methanol, with a pH adjusted to 3.2 with acetic acid. Standards were run concurrently and concentrations of unknowns were determined by
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comparison to peak areas of standards after correction for recovery of the internal standard (Leret et al., 1993). Nucleotide level determination was done in cerebellum and striatum at PN21.The samples were homogenized in cold perchloric acid 6% containing 0.8 mM EDTA, centrifuged (10 000 rpm, 10 min, 4 °C), and supernatants adjusted at pH 4.6 with KOH 6 M for the determination by HPLC with UV detection (Column Bondclone 10C18-300×3.9 mm). The mobile phase consisting of 0.2 M monopotassium phosphate adjusted to pH 6.0 with NH4OH. (Rotlla´ n et al., 1986). The total adenine nucleotides (TAN) was obtained as the sum of ATP, ADP and AMP concentrations, and the adenylate energy charge (AEC) using the formula: (ATP +1/2ADP)/TAN. Some samples were used for the estimation of ATPase activity. Cerebellum and striatum were homogenized in Tris– HCl 40 mM pH 7.4. For the total ATPase activity, the reaction was made by addition of 0.1 ml of this homogenate in 0.8 ml of medium consisting of: MgCl2 6 mM, EDTA 1 mM, NaCl 100 mM and KCl 20 mM in Tris – HCl 40 mM pH 7.4. The same medium, after addition of 1 mM ouabain, was used for the determination of Mg2 + -ATPase. The reaction was initiated by addition of 5 mM ATP, and it was stopped with cold TCA 10%. Measuring the liberated phosphorus (Pi) by colorimetric assay, the enzymatic activity was estimated. (Zaheer et al., 1968). Alkaline phosphatase activity in cerebral tissues was measured also in homogenates of cerebellum and striatum following the p-nitrophenol method (Kuftinec and Miller, 1972). Protein levels were determined (Lowry et al., 1951) for the calculation of the specific activity of the studied enzymes. Some animals, selected randomly, were maintained with distilled water ad libitum until 75 days of age, when were submitted to behavioral testing. Open-field testing was performed during a 5 min test. Exploratory activity (ambulations as number of line crossings) and rearing were noted. Behavioral results were compared by means of Mann– Whitney test after Kruskal– Wallis one-way analysis of variance (ANOVA). The results are expressed as means9standard deviation (S.D.) or means9 standard error or the
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mean (S.E.M.) (in the figures). Statistical data were analyzed using Student’s t-test, with P B 0.05.
3. Results During the treatment period, the intoxicated mothers showed a daily gain of weight equivalent to the control group. Likewise, the volume drinking and the food consumption were equal in both groups during all gestation and lactation (data not shown). The ingested dose by mothers in gestation was of 31.649 5.39 mg Pb and 1.029 0.20 mg Cd/kg weight per day. During lactation, the administered daily dose was 64.989 16.01 mg Pb and 2.179 0.87 mg Cd/ kg weight per day. The intoxicated pups showed, since birth, a lineal gain of weight statistically equal to the control pups (Fig. 1). At weaning neither the absolute or relative brain weight were affected by the metals exposure (Table 1). The Hb content in the plasma of the intoxicated group decreased significantly (8.939 1.18 g/dl in control group and 6.739 1.34 g/dl in experimental group). The hematocrit (26.59 3.74% control and 22.19 0.74% Cd–Pb group), as well as the number of red blood cells (in millions/mm3: 4.919 1.37 in control group and
Fig. 1. Weight gain in pups from birthday until postnatal day 21. Results are expressed as mean 9S.E.M. (n =8 – 11).
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Table 1 Results obtained in absolute and relative brain weight (OSI defined as brain weight×1000/body weight) and alkaline phosphatase activity in striatum and cerebellum
Brain weigth (g) Brain OSI Alkaline phosphatase activity in striatum (mmol pnitrofenol/mg prot per min) Alkaline phosphatase activity in cerebellum (mmol pnitrofenol/mg prot per min)
Control
Pb+Cd
1.3390.07 40.15 9 3.75 3.4 9 0.36
1.34 90.04 38.75 9 3.20 3.09 0.4
3.32 90.16
3.08 90.08
higher (+ 158%) in exposed animals, with a significative increase of turnover DOPAC/DA. The serotoninergic system was more affected because there was a significative increase of both 5-HT (+ 82%) and 5-HIAA ( +86%) in the cerebellum, without changes in its turnover (Table 3). The results obtained in the open-field test are showed in Fig. 5. The exposed animals present in the adulthood an increase of total as well as external ambulation, while the rearing and internal ambulation were not affected by the metals exposition.
Results are expressed as mean 9S.D. (n =8–11).
3.2091.21 in experimental group) are also reduced significantly. In conclusion, the intoxicated pups show a short-term normocitic anemia because all these parameters return to normal values at postnatal day 75 (data not shown). Fig. 2 shows the results obtained in the ATPase activity estimated in the cerebellum and striatum of pups at PN21. In both regions Pb and Cd provoked a significative reduction of Na+/K+ dependent ATPase ( −33% in cerebellum; − 23% in striatum). This effect is present only in shortterm because the activity of this enzyme was equal to the control group in both regions at PN75. Neither the total nor the Mg2 + -dependent ATPase activities were affected. The activity of the other enzyme quantified in brain, the alkaline phosphatase, was not modified for Pb and Cd intoxication neither in any regions any both ages studied (Table 1). The cerebral energetic metabolism was modified by the exposition only in the striatum of pups (Fig. 3). The ATP as well as ADP and AMP content is reduced in this area, but only the decrease of ATP was significative. Thereby, neither the AEC nor the ATP/ADP ratio were affected, but the TAN was lower than in control group (Table 2). In contrast, the monoamine contents were modified by the metals exposure only in the cerebellum (Fig. 4). The DA content was equivalent in control and treated groups, but the DOPAC was
Fig. 2. Effects of Pb and Cd on the Na+/K+-ATPase activity in Striatum (A) and Cerebellum (B) at PN21. Results are expressed as mg Pi/mg protein per h (mean 9S.E.M.), *PB 0.05, **PB 0.01 (n =8 – 11).
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Fig. 3. Nucleotide levels of Cerebellum (A) and Striatum (B) of pups at PN21. Results are expressed as mean 9S.E.M., **PB 0.01 (n =8 – 11).
4. Discussion Although heavy metals ions are known to be toxic to the central nervous system, the immature
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Fig. 4. Levels of monoamines in Cerebellum (A) and Striatum (B) of control and intoxicated pups at PN21. Results are expressed as mean 9S.E.M., *PB 0.05 (n = 9 – 10).
blood–brain barrier and the absence of protein complexes that sequester metals in mature tissues result in increased vulnerability of the fetal brain
Table 2 Total adenine nucleotides (TAN), ATP/ADP ratio and Adenylate Energy Charge (AEC) Control
Pb+Cd
Cerebellum TAN ATP/ADP AEC
146.719 24.98 1.319 0.63 0.369 0.12
158.90 956.45 1.32 90.78 0.42 90.09
Striatum TAN ATP/ADP AEC
205.83 943.74 2.03 90.81 0.42 90.09
146.94 971.59* 1.32 9 0.70 0.33 90.14
*PB0.05, Student’s t-test, results are expressed as mean 9 S.D. (n = 7–8).
Table 3 DA and 5-HIAA/5-HT ratio Control
Pb+Cd
Cerebellum DOPAC/DA 5-HIAA/5-HT
2.16 91.01 1.14 9 0.36
6.54 93.30* 1.81 90.8
Striatum DOPAC/DA 5-HIAA/5-HT
0.22 90.04 0.95 90.38
0.26 9 0.12 1.04 9 0.38
Results are expressed as mean 9S.D. (n =6–9), *PB0.05, Student’s t-test.
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Fig. 5. Results obtained in open-field test conducted at PN75 in control and intoxicated animals. Results are expressed as mean9 S.E.M., *P B0.05 (n=9–10).
to lead and cadmium. In this sense, our results confirm that Pb and Cd may exert an inhibitory effect directly on Na+/K+-ATPase activity. This enzyme plays a vital role in linking the extracellular signals to the intracellular medium in neural tissues, so the inhibition of this enzyme by Cd and Pb could be damaging to the brain cells leading to an earlier stage of edema, followed by a later stage of degeneration and necrosis. Several previous works have shown that perinatal exposition to these metals, even at doses lower than here, can inhibit the synaptosomal Na+/K+-ATPase (Rafalowska et al., 1996). Pb and Cd could bind to free SH groups and inhibit reversibly the enzyme, so that mechanism could explain why isoforms with subunits a2 and a3, limited to nervous tissue and cardiac muscle, are more susceptible to the inactivation for their higher content of cysteins (Fox et al., 1991). On the other hand, the in vitro studies indicates that Cd is a more potent inhibitor than Pb, related to its high affinity for the free SH (Carfagna et al., 1996). In our laboratory, we found also in cortex and cerebellum a significant inhibition of this enzyme after perinatal exposure to isolated Pb, although the rate of inactivation was lower than in this work (Antonio and Leret, 2000), suggesting an additive effect, in agreement with the in vitro studies (Carfagna et al., 1996). Alkaline phosphatase has been recently implicated in neuritogenesis processes, even in early
development, and also in mielinization processes, which in rats occurs rapidly in the first 2 weeks after birth. Likewise, it could participate on energetic metabolism and on transduction signal pathways (Kern and Audesirk, 1995; Shinozaki and Pritzker, 1996). A similar exposition to Pb in our laboratory, provoked a fall on its cerebral activity at PN12 (Antonio and Leret, 2000). This enzyme is Zn-dependent, so depletion on cerebral Zn caused by Pb and Cd (Gupta et al., 1993) and/or their binding to SH groups, could explain its inhibition. In our study no effects have been detected at this level, which could be related to the age of the animals, because the inhibition cited above coincided with the intense mielinization period, or with a possible antagonic effects between both metals. With respect to brain nucleotide levels, a decrease was observed in this study only in the striatum of exposed pups. In previous works conducted in our laboratory we observed that using lead alone intoxication protocol, striatum was also more affected (Antonio and Leret, 2000). Pb has been proposed to inhibit the oxidative phosphorilation, although the exact target is still unclear. Some authors maintain that, since synaptosomal respiration results unaltered after Pb poisoning, ATP depletion could be related to a structural and functional damage of mitochondrial membranes (Rafalowska et al., 1996), or to a delayed maturation of oxidative metabolism (Bull, 1983). Cadmium toxicity implicates the inhibition of the respiratory chain, both on I and II complexes (Miccadei and Floridi, 1993). Alteration of cerebral monoamines content has been frequently related to these heavy metals poisoning. Although the effects provoked by Cd are not fully understood, doses similar to ours are associated with an increase of monoamine levels (Ma´ rquez et al., 1999). Pb seems to have opposite effects, causing a drop in similar expositions. This antagonic effect has been contrasted in our laboratory by individual administration of the same doses of these metals (Antonio et al., 1996, 1998). Alteration in neurotransmitters release mechanisms, or in their synthesis and catabolism, seems to participate on these effects. Cd is able to stimulate cathecolamine synthesis, by activating
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the calmodulin dependent pathway, and even inhibiting catabolic enzymes, such as monoaminoxidases (Leung et al., 1992). In contrast, Pb could interact with some signal pathways, which are implicated in monoamine synthesis, as well as with tyrosine hydroxylase activity or the tetrahydrobiopterin synthesis (Ramin and Porter, 1997). Cotreatment with cadmium and lead difficult the interpretation of the results. The increase of indolamines and DOPAC in the cerebellum of intoxicated pups does not belong with the results obtained from perinatal administration of Pb at postnatal days 0, 5 and 12, where a decrease on monoamines content were observed (Antonio et al., 1996; Antonio and Leret, 2000). Our results are more comparable to those produced by Cd alone (Antonio et al., 1998). Two stages could be distinguished during the exposition period, according to the bioavailability of these metals. Throughout pregnancy, the availability of lead is higher, while during lactation takes place the highest capture and storage of Cd by the pups. Perhaps we are detecting the combined effects, although, due to their recent exposition, those related to Cd are more evident in the brain of the pups. Excepting the change in ATP levels, the cerebellum has been more sensitive to intoxication. It has been described the homogeneity of these metals in various regions of the brain (Widzowski and Cory-Sletcha, 1994)), so its higher sensitivity could be due to functional features. Thus, differences in both regions development must be considered, because this study was conducted in this critic period. Most of the cells appear in striatum during fetal life, when Cd exposure is lower, while the cerebellum development extends until advanced lactation, when both metals are fully available, and its higher susceptibility could be due to the combined exposition to lead and cadmium. The effects of lead and cadmium on neurobehavioral development have been extensively investigated in humans. Locomotion and conditioned responses dysfunctions, as well as decreased intelligence, hyperactivity, reduced learning/memory capacities and deficient IQ scores are the main symptoms observed in both children and adults exposed to lead and/or cadmium (Taylor and
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Ennever, 1993; Tong, 1998). In animals, behavioral effects of Cd are very controversial, but are normally associated with a decrease in ambulation and rearing in open-field test (De´ si et al., 1998). However, our results are similar to the effects described after lead poisoning (Ma, et al., 1999), and the interaction with dopaminergic system has been often involved in this hyperactivity (CorySletcha, 1995; Ma, et al., 1999). Morphological and electrophysiological alterations in cerebellum could be also underlying, such as molecular layer atrophy and decrease in granulate cells density and spontaneous discharge of Purkinje cells (Bo¨ rjklund et al., 1983). On the other hand, Pb could alter synaptic plasticity by interaction with adhesion molecules during early development (Murphy and Regan, 1999), which could explain the persistence of effects even in adult life. In conclusion, we have detected after perinatal exposure to low to moderate doses of Pb and Cd, neurochemical alterations in cerebellum and striatum at weaning, such as the decrease in enzymes activities and energetic metabolism, or the alteration in the monoamines content. An important finding in this study is that these effects could be responsible of the alterations in motor behavior observed in the adulthood of intoxicated rats.
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