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Study of the neurochemical alterations produced in discrete brain areas by perinatal low-level lead exposure
Life Sciences 67 (2000) 635Ð642
Study of the neurochemical alterations produced in discrete brain areas by perinatal low-level lead exposure M.T. Antonio*, M.L. Leret Department of Animal Biology II, Fac. Biology, Complutense University, 20840 Madrid, Spain
Abstract Although the neurotoxic effects of Pb are well documented, the subcellular mechanisms of this action in the central nervous system are not fully understood. The present work examines some neurochemical parameters in discrete brain areas of pups whose mothers were intoxicated via drinking water with lead (300 mg/L), from day 1 of pregnancy until postnatal day 12. Lead intoxication produced a signiÞcant reduction in the activity of the enzymes alkaline phosphatase and ATP-ase in the brain. Furthermore, the levels of adenine nucleotides were signiÞcantly altered by treatment, the striatum being the area more affected, whereas lead did not vary the levels of ATP, ADP and AMP in the hypothalamus. On the other hand, there was a general decrease in neurotransmitter levels in all areas, specially in the hippocampus. These data suggest that gestational and lactational exposure to low dose of lead could produce neurochemical changes in discrete brain areas which can be responsible for the neurophysiological and behavioral changes described in lead-intoxicated animals. © 2000 Elsevier Science Inc. All rights reserved. Keywords: Lead; Alkaline phosphatase; Adenosine triphosphatase; Monoamines; Nucleotides triphosphate
Introduction Lead toxicity remains a signiÞcant public health problem, since blood Pb levels as low as 10 mg/dL have been shown to cause signiÞcant impairment of cognitive functioning and signiÞcant delays in behavioral development in children (1). Thus, a continued effort has been made to deÞne the effects of Pb on central nervous system function in animals when exposure occurs during development. Rapid changes in organ development and function occurs during the neonatal period, because the central nervous system is in rapid growth rate and highly vulnerable to the toxic effects of lead. This metal passes through the placenta, and maternal and fetal blood levels
* Corresponding author. Fax: 34-91-394-4935. E-mail address: [email protected] (M.T. Antonio) 0024-3205/00/$ Ð see front matter © 2000 Elsevier Science Inc. All rights reserved. PII: S 0 0 2 4 - 3 2 0 5 ( 0 0 )0 0 6 5 5 -X
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are highly correlated (2). Lead is also excreted into milk during the lactational period (3). The immature blood-brain barrier and the absence of protein complexes that sequester lead in mature tissues result in increased vulnerability of the fetal brain to lead (4). Due to their high gastrointestinal absorption and less effective renal excretion, the tissue levels of lead can reach higher levels in neonates than in adults. Most of the neurotoxic metals, including Pb, are believed to be non-speciÞc toxicants that react with a wide spectrum of cellular components, disturbing many cellular functions. At molecular level, among other biochemical effects, lead can inhibit free sulphydryl groupdependent enzymes, and alter cerebral metabolism, membrane permeability and energy production (5,6). This metal competes with calcium, inhibiting the release of neurotransmitters and interferes with the regulation of cell metabolism by binding to second messenger calcium receptors, blocking calcium transport by calcium channels and calcium-sodium ATP pumps, and by competing for calcium-binding protein sites and uptake by mitochondria (7,8). The aim of the experiments presented here is to discern whether prolonged consumption of leaden water during gestation and lactational period affects some neurochemical parameters in discrete brain areas; when the central nervous system is characterized by an intensive process of cellular proliferation and differentiation. Material and methods Time-pregnant rats from a laboratory-inbred Wistar strain were maintained on a 12-hour light/dark cycle and fed daily with a special diet for rat gestational stages. Groups of four dams were administered lead acetate (300 mg/L dissolved in distilled water) ad libitum or distilled water from the beginning of pregnancy to day 12 after parturition. Through the experimental period, maternal water consumption and body weight gain were measured daily. At birthday litters were culled to 12Ð15 pups, and their body weight gain was also measured daily. At postnatal day 12, pups were sacriÞced. Blood samples were taken immediately after the sacriÞce of animals and hematocrit value was determined by standard hematological techniques. Hemoglobin content was determined by cyanmethemoglobin method (9). Lead was measured in blood using a graphite furnace atomic absorption spectrophotometer (Spectra AA-10/20, Varian) after wet digestion of the samples with a mixture of concentrated perchloric and nitric acids (10). Glucose was also measured in plasma by a commercial kit. After sacriÞce, the whole brains were quickly (,45 s) removed and frozen at 2808C. A series of 1.0Ð1.5 mm-thick sections were cut on a cold plate , and the hippocampus, striatum, hypothalamus and cerebellum quickly dissected , as previously described (11). These areas were used for the determination of neurotransmitters and nucleotides, whereas the cerebral cortex was used for the determination of cholesterol levels (12), alkaline phosphatase activity (13), as well as ATP-ase activity (14). Some encephalons, selected randomly, were used for brain monoamines determination (15). For this purpose, samples were homogenized in 0.5 ml of cold 0.2 N perchloric acid containing 0.4 mM sodium bisulphite and 0.4 mM EDTA. 3,4-dihydroxybenzylamine was also added to each sample as an internal standard to control for procedural losses. The homogenates were centrifuged (13.000 rpm, 5 min. at 48C) and supernatants used for determination of levels of noradrenaline (NA), dopamine (DA), 3,4-dihydroxyphenilacetic acid (DOPAC),
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5-hydroxytryptamine (5-HT) and 5-hydroxyindolacetic acid (5-HIAA) using high performance liquid chromatography with electrochemical detection (HPLC-ED) (Column Spherisorb ODS2 (22 cm 3 4.6 mm) after precolumn 10 glc 4 ODS2, then eluted with a mobile phase consisting of 0.05 M monopotassium phosphate, 0.1 mM EDTA, 1 mM heptane sulphonate and 8% methanol (pH adjusted to 3.2 with acetic acid). Standards were run concurrently and concentrations of unknowns were determined by comparison to peak areas of standards after correction for recovery of the internal standard. For determination of nucleotides levels (16), samples were homogenized in 1 ml of cold 6% perchloric acid containing 0.8 mM EDTA. Homogenates were centrifuged (10,000 rpm, 10 min. at 48C) and supernatants were adjusted to pH 4.6 with 6 M KOH for determination of ATP, ADP and AMP levels by high performance liquid chromatography with UV detection (HPLC-UV), [column Bondclone 10C 18 (300 3 3.9 mm)], then eluted with a mobile phase consisting of 0.2 M monopotassium phosphate (pH adjusted to 6 with NH4OH). ATP, ADP and AMP were summed to obtain total adenine nucleotides (T.A.N.), whereas the adenylate energy charge (A.E.C.) was calculated using the formula: (ATP 11/21ADP)/T.A.N. ATP-ase activity from control and lead-exposed rats was measured spectrophotometrically by determining the inorganic phosphate (Pi) liberated. Samples were homogenized in TrisClH pH 7.4. ATP-ase activity of 0.1 ml aliquots of the homogenates was measured in a Þnal volume of 1 ml containing 40 mM Tris-ClH, 100 mM NaCl, 6 mM MgCl2, 20 mM KCl, 1 mM EDTA and the presence or absence of 1 mM ouabain. The reaction was stopped at 10 min. by the addition of ice-cold 10% trichloroacetic acid, samples were centrifuged at 1500 g for 10 min, and the Pi was determined colorimetrically in the protein-free supernatant. Na1/K1ATP-ase activity was calculated as the difference between the total ATP-ase activity (samples without ouabain) and Mg21-ATP-ase activity (samples with ouabain). Protein was determined by the method of Lowry et al. (17) using bovine serum albumin as standard. Results are expressed as mean 6 standard deviation (SD) or mean 6 standard error of the mean (SEM) (In Þgures). StudentÕs t-test for control and experimental groups was used for statistical free comparison of the different parameters studied, p , 0.05 was considered to be signiÞcant.
Fig. 1. Weight gain in pups from birthday until postnatal day 12. Results are expressed as mean. n540Ð48. ** p , 0.01.
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Table 1 Hematological parameters and lead levels in pupÕs blood Hemoglobin (g/dL) Glucose (mg/dL) Hematocrit (%) Lead (mg/dL)
Lead
Control
8.56 6 1.49 89.23 6 22.97 26.38 6 0.52 29.32 6 8.15
7.85 6 1.31 98.89 6 20.70 30.20 6 1.30** 6.57 6 2.54**
n512Ð20. ** p , 0.01.
Results The amount of Pb ingested during the experimental period (33 days) was 35.92 6 12.01 mg/Kg body weight-day. It was calculated using the formula: (mL water consumed/day 3 lead dose)/rat weight (kg). No effects were observed on body weight gain or water consumption of dams (data not shown). At parturition, there were not differences between the weight of control and experimental pups, but during lactation, these differences were signiÞcant from day 6 until day 12 postnatal (Fig. 1). Although blood lead levels in treated pups were signiÞcantly higher than controls, hemoglobin and plasma glucose levels were not affected by the treatment, but we observed a signiÞcant decrease in the hematocrit value (Table 1). In the brain, lead exposure did not affect the cholesterol levels, but produced a signiÞcant reduction of brainÕs weight and alkaline phosphatase activity (Table 2). Na1/K1 ATP-ase activity decreased in the cerebral cortex of pups intoxicated with lead, whereas in cerebellum both ATP-ases (Na1/K1 and Mg21-dependent) activities decreased signiÞcantly (Figs. 2a and 2b). On the other hand, the energy metabolism of discrete brain areas was signiÞcantly altered by lead-treatment of rats. The more affected area was the striatum, where ATP, ADP and AMP levels decreased signiÞcantly in the intoxicated pups. However, lead did not vary the levels of adenine nucleotides in hypothalamus (See Figs, 3a, 3b, 3c and 3d). Adenylate energy charge, an additional index of cell energy state, was decreased also in the striatum of intoxicated pups (225%).Moreover, lead treatment diminished signiÞcantly the ATP/ADP ratio in striatum and hippocampus (see also Figs. 3aÐd). Data presented here show that signiÞcant changes in neurotransmitter levels occur in several brain areas of lead-treated rats. The most affected area was the hippocampus, where DA, 5-HT as well as their main respective metabolites, DOPAC and 5-HIAA decreased signiÞ-
Table 2 Brain weight, cholesterol levels and alkaline phosphatase activity in brains of control and lead-treated pups Weight (mg) Cholesterol (mg/g ) Phosphatase alkaline (UI/g) n512Ð20. ** p , 0.01.
Lead
Control
923.57 6 27.35 8.28 6 3.66 14.55 6 2.92
986.67 6 46.59** 10.07 6 3.84 17.70 6 2.40**
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Fig. 2. Effects of Pb on the activity of Na1/K1 ATP-ase (A) and Mg21 ATP-ase (B) in cerebral cortex (Fig. 2a) and cerebellum (Fig. 2b). Results are expressed as mM Pi/mgprot./hour (mean 6 s.e.m.) n511Ð12. * p , 0.05 ** p , 0.01.
cantly (Fig. 4a). In the hypothalamus (Fig. 4b), NA, DA and 5-HT decreased signiÞcantly, whereas in the cerebellum of lead-treated rats, NA and 5-HT showed a signiÞcant reduction (277.71% and 223.56% respectively) (Fig. 4c). The striatum was the area less affected, since only DA levels showed a strong reduction (231.65%) in the intoxicated pups (Fig. 4d).
Fig. 3. Nucleotide levels of different brain areas (Fig. 3a hippocampus, 3b hypothalamus, 3c cerebellum and 3d striatum. Results are expressed as mean 6 s.e.m.. (n5 7Ð10) * p , 0.05 ** p , 0.01. Control u; Lead j
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Fig. 4. Levels of neurotransmitters in hippocampus (4a), hypothalamus (4b), cerebellum (4c) and striatum (4d) of control and intoxicated pups. Results are expressed as mean 6 s.e.m. (n57Ð10) * p , 0.05 ** p , 0.01.
Discussion The changes we observed were not accompanied by signiÞcant general toxic effects, because none of the investigated dams growth parameters revealed any signiÞcant difference between control animals and lead-treated ones. These results are in concordance with previous Þndings of this and similar lead dosing models (18Ð20). In pups, although a signiÞcant reduction in body weight was found since postnatal day 6, no differences were detected in hemoglobin and plasma glucose levels, so it seems that the lead treated pups have a good physiological status. The decrease in the values of hematocrit conÞrm the observation that lead exposure can produce hematological disturbances, including poikilocytosis, perhaps due to the oxidative stress induced by lead in red blood cells (5). Since the blood/brain barrier does not form a substantial impediment to lead entry into the developing brain, the brain lead levels (mg/100g) are about 1Ð3 times the concentration in whole blood (mg/100 ml) (21). Although we had not measured the lead concentration in the brain, we expect neurochemical changes due to lead exposure. We have measured alkaline phosphatase activity (ALP), which is a zinc metalloenzyme involved in both maturation and myelination. The decrease of this enzyme activity could be related to the decrease of zinc concentration described by several authors in lead intoxication (22). Recent studies have suggested that ALP might have more global biological functions because ALP from diverse sources has been shown to have pyrophosphatase activity and
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phosphate ester hydrolysis in biological systems has become recognized as an important process linking energy metabolism and cell signal transduction pathways (23). We also conÞrmed that Pb may exert an inhibitory effect directly on Na1/K1ATP-ase activity in cerebellum and cerebral cortex of intoxicated pups. It is known that Na1/K1ATPase, a key enzyme for maintaining the ion distribution about the cellular membrane which is required for neuronal activity, is among the enzymes particularly affected by lead (24,25). This fact may relate to the different alpha subunit composition of Na1/K1ATP-ase of different tissues. Enzyme extracted from brain has mostly a2 an a3 isozymes present in signiÞcant amounts (26) All the subunits of the enzyme (a1, 2 and 3) differ with the number of reactive sulphydryl groups in the catalytic site (27) and a2 and 3 forms of the enzyme are much more sensitive to the inhibitory effect of Pb than the a1 form is (24). Thus it can be suggested that one of the possible mechanisms of the inhibition may be the interaction of Pb with -SH groups in the catalytic site of the enzyme. The general decrease in neurotransmitters observed in our study could be related with a decreased synthetic capacity, because it has been described that lead could inhibit tyroxine hydroxylase activity (28) or alternatively with lead interference with cellular energy metabolism, inhibiting ATP synthesis that leads to the dysfunction of energy-dependent neuronal events such as neurotransmitter uptake (6). There was not an homogeneous response within the different brain areas. This agrees with others studies where it has been observed that lead, depending on the dose has disparate effects on the neurotransmitter levels in a given region of the brain and that the concentrations of neurotransmitters in various regions of the brain responded differently to the same dose of Pb21. In this sense, previous studies performed in our laboratory, with similar doses of lead acetate, showed normal 5-HT values in presence of increased 5-HIAA on postnatal day 0 and reduced 5-HT with reduced 5-HIAA values on postnatal day 5 (20). Likewise, it has been described (18) in a pre- and postnatal lead exposure that produced blood levels of 50Ð60 mg/100 mL, a signiÞcant reduction of 5-HT in brain stem, whereas 5-HIAA was reduced both in the brain stem and striatum. However, when lead was administrated in adult rats, although the blood levels reached were similar, only the hypothalamus showed a signiÞcant increase in DOPAC levels, whereas the rest of brain areas studied (striatum, hippocampus, midbrain and cortex) did not show changes in monoamine levels (29). This could not be related with a preferential accumulation of lead, since it has been described a homogeneous distribution of lead within the brain (19), but with local differences in the formation and maturation of the different brain regions and transmitter identiÞed systems. Monoamine tissue levels usually provide a good estimate of the innervation density (30), and these results indicate that the monoamine Þbers (i.e. the synaptogenesis) were affected by the gestational and lactational lead exposure and it could be reßected as later behavioral changes. In this sense, the hippocampus was chosen as the anatomical site for learning and memory and as an important target of neurotoxic agents. The neuroanatomical and functional development of the rat hippocampus extend up to the second postnatal week. The experimental paradigm of lead exposure was designated to include this period. In fact, this area was the most affected in relation to the decrease in neurotransmitters as well as the diminution of energy charge. This fact can be related with the impairment of long-term potentiation and learning following perinatal lead exposure described in the literature (31). Likewise, the disturbed
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striatal and cerebellar functions may contribute to the spectrum of symptoms such as hyperactivity and motor coordination problems associated with lead exposure (1). In our work, the chronic pre- and postnatal exposure of rats to low levels of lead via dams resulted in blood concentrations in the offspring of about 29 mg/dL, a value only slightly higher than levels found in children environmentally exposed to this pollutant. This in turn points to the need to deÞne and follow behavioral and neurophysiological biomarkers of lowlevel lead exposure. References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31.
N. L. NEEDLEMAN (Ed). Human Lead Exposure. CRC Press. 1992. K. N. DIETRICH. Fund. Appl. Toxicol. 16 17Ð19 (1990). H. I. PALMINGER and A. OSKARSSON. Pharmacol. Toxicol., 73 174Ð179 (1993). R. A. GOYER. Environ. Health Perspect., 89 101Ð105 (1990). H. G†RER, H.…ZG†NES, R. NEAL, D. R. SPITZ and N. ER‚AL. Toxicology, 128 181Ð189 (1998). M. J. BOYKIN, C. S. CHETTY and B. RAJANNA. Ecotoxicol. Environ. Saf., 22 88Ð93 (1991). J. LUTHMAN, E. LINDQVIST, G. A. GERHARDT, L. OLSON and B. H. HOFFER. Environ. Res., 65 100Ð 118 (1994). R. BETTAIYA, P. R. YALLAPRAGADA, E. HALL and S. RAJANNA. Ecotoxicol. Environ. Saf., 33 157Ð 162 (1996). J. E. KAMPEN and G. W. ZIJLSTRA. Clin. Chem. Acta, 6 538Ð545 (1961). M. T. ANTONIO and I. CORPAS. Environ. Technol. Lett., 9 227Ð238 (1988). F. MOLINA-HOLGADO, E. MOLINA-HOLGADO, M. L. LERET, I. GONZALEZ and T. A. READER. J. Neurochem. Res., 18 1183Ð1191 (1993). W. D. BLOCK, K. J. JARRET and J. B. LEVINE. Clin. Chem., 12 681Ð689 (1966). M. M. KUFTINEC and S. A. MILLER. Calcif. Tissue Res., 9 173Ð178 (1972). N. N. ZAHEER, Z. IQBAL and G. P. TALWAR. J. Neurochem., 15 1217Ð1224 (1968). M. L. LERET, M. I. GONZALEZ and R. ARAHUETES. Life Sci.,52 1609Ð1615 (1993). J. WYNNANTS and H. VAN BELLE. Anal. Biochem., 144 258Ð266 (1985). O. H. LOWRY, N. J. ROSEBROUGH, A. L. FARR and R. J. RANDAL. J. Biol. Chem., 193 265Ð275 (1951). H. R. WIDMER, E. E. B†TIKOFER, M. SCHLUMPF and W. LICHTENSTEIGER. Experientia, 47 463Ð 466 (1991). D. W. WIDZOWSKI and D. A. CORY-SLECHTA. Neurotoxicology, 15 295Ð308 (1994). M. T. ANTONIO, S. MARTINEZ, M. L. LERET and I. CORPAS. J Appl. Toxicol., 16 431Ð436 (1996). D. A. CORY-SLECHTA. Toxicol. Appl. Pharmacol., 104 67Ð78 (1990). F. A. ADENIYI and F. W. HEATON. Br. J. Rheumatol., 43 561Ð569 (1980). T. SHINOZAKI and K. P. H. PRITZER. J. Rheumatol., 23 677Ð683 (1996). D. A. FOX, S. D. RUBINSTEIN and P. HSU. Toxicol. Appl. Pharmacol., 109 482Ð493 (1991) M. A. CARFAGNA, G. D. PONSLER and B. B. MUHOBERAC. Chem. Biol. Interactions, 100 53Ð65 (1996). A. GERBI, M. DEBRAY, J. M. MAIXENT, C. CHANEZ and J. M. BOURRE. J.Neurochem., 60 247Ð252 (1993). J. L. BRODSKY and G. GUIDOTTI. An. Physiol. Soc., C803ÐC811 (1990). S. M. RAMIN, W. KEDZIERSKI and J. C. PORTER. Mol. Cell Neurosci., 4 449Ð454 (1993). J. J. MEJIA, F. DIAZ-BARRIGA, J. CALDERON, C. RIOS, M. E. JIMENEZ-CAPDEVILLE. Toxicol. Teratol. 19 489Ð497 (1997). J. LUTHMAN, E. BRODIN, E. SUNDSTR…M and B. WIEHAGER. Int. J. Dev. Neurosci., 8 549Ð560 (1990). L. ALTMANN, F. WEINSBERG, K. SUEISSON, H. LILIENTHAL, K. WIEGAND, G. WINNWKE. Toxicol. Lett. 66 105Ð112 (1993).
Study of the neurochemical alterations produced in discrete brain areas by perinatal low-level lead exposure M.T. Antonio*, M.L. Leret Department of Animal Biology II, Fac. Biology, Complutense University, 20840 Madrid, Spain
Abstract Although the neurotoxic effects of Pb are well documented, the subcellular mechanisms of this action in the central nervous system are not fully understood. The present work examines some neurochemical parameters in discrete brain areas of pups whose mothers were intoxicated via drinking water with lead (300 mg/L), from day 1 of pregnancy until postnatal day 12. Lead intoxication produced a signiÞcant reduction in the activity of the enzymes alkaline phosphatase and ATP-ase in the brain. Furthermore, the levels of adenine nucleotides were signiÞcantly altered by treatment, the striatum being the area more affected, whereas lead did not vary the levels of ATP, ADP and AMP in the hypothalamus. On the other hand, there was a general decrease in neurotransmitter levels in all areas, specially in the hippocampus. These data suggest that gestational and lactational exposure to low dose of lead could produce neurochemical changes in discrete brain areas which can be responsible for the neurophysiological and behavioral changes described in lead-intoxicated animals. © 2000 Elsevier Science Inc. All rights reserved. Keywords: Lead; Alkaline phosphatase; Adenosine triphosphatase; Monoamines; Nucleotides triphosphate
Introduction Lead toxicity remains a signiÞcant public health problem, since blood Pb levels as low as 10 mg/dL have been shown to cause signiÞcant impairment of cognitive functioning and signiÞcant delays in behavioral development in children (1). Thus, a continued effort has been made to deÞne the effects of Pb on central nervous system function in animals when exposure occurs during development. Rapid changes in organ development and function occurs during the neonatal period, because the central nervous system is in rapid growth rate and highly vulnerable to the toxic effects of lead. This metal passes through the placenta, and maternal and fetal blood levels
* Corresponding author. Fax: 34-91-394-4935. E-mail address: [email protected] (M.T. Antonio) 0024-3205/00/$ Ð see front matter © 2000 Elsevier Science Inc. All rights reserved. PII: S 0 0 2 4 - 3 2 0 5 ( 0 0 )0 0 6 5 5 -X
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are highly correlated (2). Lead is also excreted into milk during the lactational period (3). The immature blood-brain barrier and the absence of protein complexes that sequester lead in mature tissues result in increased vulnerability of the fetal brain to lead (4). Due to their high gastrointestinal absorption and less effective renal excretion, the tissue levels of lead can reach higher levels in neonates than in adults. Most of the neurotoxic metals, including Pb, are believed to be non-speciÞc toxicants that react with a wide spectrum of cellular components, disturbing many cellular functions. At molecular level, among other biochemical effects, lead can inhibit free sulphydryl groupdependent enzymes, and alter cerebral metabolism, membrane permeability and energy production (5,6). This metal competes with calcium, inhibiting the release of neurotransmitters and interferes with the regulation of cell metabolism by binding to second messenger calcium receptors, blocking calcium transport by calcium channels and calcium-sodium ATP pumps, and by competing for calcium-binding protein sites and uptake by mitochondria (7,8). The aim of the experiments presented here is to discern whether prolonged consumption of leaden water during gestation and lactational period affects some neurochemical parameters in discrete brain areas; when the central nervous system is characterized by an intensive process of cellular proliferation and differentiation. Material and methods Time-pregnant rats from a laboratory-inbred Wistar strain were maintained on a 12-hour light/dark cycle and fed daily with a special diet for rat gestational stages. Groups of four dams were administered lead acetate (300 mg/L dissolved in distilled water) ad libitum or distilled water from the beginning of pregnancy to day 12 after parturition. Through the experimental period, maternal water consumption and body weight gain were measured daily. At birthday litters were culled to 12Ð15 pups, and their body weight gain was also measured daily. At postnatal day 12, pups were sacriÞced. Blood samples were taken immediately after the sacriÞce of animals and hematocrit value was determined by standard hematological techniques. Hemoglobin content was determined by cyanmethemoglobin method (9). Lead was measured in blood using a graphite furnace atomic absorption spectrophotometer (Spectra AA-10/20, Varian) after wet digestion of the samples with a mixture of concentrated perchloric and nitric acids (10). Glucose was also measured in plasma by a commercial kit. After sacriÞce, the whole brains were quickly (,45 s) removed and frozen at 2808C. A series of 1.0Ð1.5 mm-thick sections were cut on a cold plate , and the hippocampus, striatum, hypothalamus and cerebellum quickly dissected , as previously described (11). These areas were used for the determination of neurotransmitters and nucleotides, whereas the cerebral cortex was used for the determination of cholesterol levels (12), alkaline phosphatase activity (13), as well as ATP-ase activity (14). Some encephalons, selected randomly, were used for brain monoamines determination (15). For this purpose, samples were homogenized in 0.5 ml of cold 0.2 N perchloric acid containing 0.4 mM sodium bisulphite and 0.4 mM EDTA. 3,4-dihydroxybenzylamine was also added to each sample as an internal standard to control for procedural losses. The homogenates were centrifuged (13.000 rpm, 5 min. at 48C) and supernatants used for determination of levels of noradrenaline (NA), dopamine (DA), 3,4-dihydroxyphenilacetic acid (DOPAC),
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637
5-hydroxytryptamine (5-HT) and 5-hydroxyindolacetic acid (5-HIAA) using high performance liquid chromatography with electrochemical detection (HPLC-ED) (Column Spherisorb ODS2 (22 cm 3 4.6 mm) after precolumn 10 glc 4 ODS2, then eluted with a mobile phase consisting of 0.05 M monopotassium phosphate, 0.1 mM EDTA, 1 mM heptane sulphonate and 8% methanol (pH adjusted to 3.2 with acetic acid). Standards were run concurrently and concentrations of unknowns were determined by comparison to peak areas of standards after correction for recovery of the internal standard. For determination of nucleotides levels (16), samples were homogenized in 1 ml of cold 6% perchloric acid containing 0.8 mM EDTA. Homogenates were centrifuged (10,000 rpm, 10 min. at 48C) and supernatants were adjusted to pH 4.6 with 6 M KOH for determination of ATP, ADP and AMP levels by high performance liquid chromatography with UV detection (HPLC-UV), [column Bondclone 10C 18 (300 3 3.9 mm)], then eluted with a mobile phase consisting of 0.2 M monopotassium phosphate (pH adjusted to 6 with NH4OH). ATP, ADP and AMP were summed to obtain total adenine nucleotides (T.A.N.), whereas the adenylate energy charge (A.E.C.) was calculated using the formula: (ATP 11/21ADP)/T.A.N. ATP-ase activity from control and lead-exposed rats was measured spectrophotometrically by determining the inorganic phosphate (Pi) liberated. Samples were homogenized in TrisClH pH 7.4. ATP-ase activity of 0.1 ml aliquots of the homogenates was measured in a Þnal volume of 1 ml containing 40 mM Tris-ClH, 100 mM NaCl, 6 mM MgCl2, 20 mM KCl, 1 mM EDTA and the presence or absence of 1 mM ouabain. The reaction was stopped at 10 min. by the addition of ice-cold 10% trichloroacetic acid, samples were centrifuged at 1500 g for 10 min, and the Pi was determined colorimetrically in the protein-free supernatant. Na1/K1ATP-ase activity was calculated as the difference between the total ATP-ase activity (samples without ouabain) and Mg21-ATP-ase activity (samples with ouabain). Protein was determined by the method of Lowry et al. (17) using bovine serum albumin as standard. Results are expressed as mean 6 standard deviation (SD) or mean 6 standard error of the mean (SEM) (In Þgures). StudentÕs t-test for control and experimental groups was used for statistical free comparison of the different parameters studied, p , 0.05 was considered to be signiÞcant.
Fig. 1. Weight gain in pups from birthday until postnatal day 12. Results are expressed as mean. n540Ð48. ** p , 0.01.
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Table 1 Hematological parameters and lead levels in pupÕs blood Hemoglobin (g/dL) Glucose (mg/dL) Hematocrit (%) Lead (mg/dL)
Lead
Control
8.56 6 1.49 89.23 6 22.97 26.38 6 0.52 29.32 6 8.15
7.85 6 1.31 98.89 6 20.70 30.20 6 1.30** 6.57 6 2.54**
n512Ð20. ** p , 0.01.
Results The amount of Pb ingested during the experimental period (33 days) was 35.92 6 12.01 mg/Kg body weight-day. It was calculated using the formula: (mL water consumed/day 3 lead dose)/rat weight (kg). No effects were observed on body weight gain or water consumption of dams (data not shown). At parturition, there were not differences between the weight of control and experimental pups, but during lactation, these differences were signiÞcant from day 6 until day 12 postnatal (Fig. 1). Although blood lead levels in treated pups were signiÞcantly higher than controls, hemoglobin and plasma glucose levels were not affected by the treatment, but we observed a signiÞcant decrease in the hematocrit value (Table 1). In the brain, lead exposure did not affect the cholesterol levels, but produced a signiÞcant reduction of brainÕs weight and alkaline phosphatase activity (Table 2). Na1/K1 ATP-ase activity decreased in the cerebral cortex of pups intoxicated with lead, whereas in cerebellum both ATP-ases (Na1/K1 and Mg21-dependent) activities decreased signiÞcantly (Figs. 2a and 2b). On the other hand, the energy metabolism of discrete brain areas was signiÞcantly altered by lead-treatment of rats. The more affected area was the striatum, where ATP, ADP and AMP levels decreased signiÞcantly in the intoxicated pups. However, lead did not vary the levels of adenine nucleotides in hypothalamus (See Figs, 3a, 3b, 3c and 3d). Adenylate energy charge, an additional index of cell energy state, was decreased also in the striatum of intoxicated pups (225%).Moreover, lead treatment diminished signiÞcantly the ATP/ADP ratio in striatum and hippocampus (see also Figs. 3aÐd). Data presented here show that signiÞcant changes in neurotransmitter levels occur in several brain areas of lead-treated rats. The most affected area was the hippocampus, where DA, 5-HT as well as their main respective metabolites, DOPAC and 5-HIAA decreased signiÞ-
Table 2 Brain weight, cholesterol levels and alkaline phosphatase activity in brains of control and lead-treated pups Weight (mg) Cholesterol (mg/g ) Phosphatase alkaline (UI/g) n512Ð20. ** p , 0.01.
Lead
Control
923.57 6 27.35 8.28 6 3.66 14.55 6 2.92
986.67 6 46.59** 10.07 6 3.84 17.70 6 2.40**
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Fig. 2. Effects of Pb on the activity of Na1/K1 ATP-ase (A) and Mg21 ATP-ase (B) in cerebral cortex (Fig. 2a) and cerebellum (Fig. 2b). Results are expressed as mM Pi/mgprot./hour (mean 6 s.e.m.) n511Ð12. * p , 0.05 ** p , 0.01.
cantly (Fig. 4a). In the hypothalamus (Fig. 4b), NA, DA and 5-HT decreased signiÞcantly, whereas in the cerebellum of lead-treated rats, NA and 5-HT showed a signiÞcant reduction (277.71% and 223.56% respectively) (Fig. 4c). The striatum was the area less affected, since only DA levels showed a strong reduction (231.65%) in the intoxicated pups (Fig. 4d).
Fig. 3. Nucleotide levels of different brain areas (Fig. 3a hippocampus, 3b hypothalamus, 3c cerebellum and 3d striatum. Results are expressed as mean 6 s.e.m.. (n5 7Ð10) * p , 0.05 ** p , 0.01. Control u; Lead j
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Fig. 4. Levels of neurotransmitters in hippocampus (4a), hypothalamus (4b), cerebellum (4c) and striatum (4d) of control and intoxicated pups. Results are expressed as mean 6 s.e.m. (n57Ð10) * p , 0.05 ** p , 0.01.
Discussion The changes we observed were not accompanied by signiÞcant general toxic effects, because none of the investigated dams growth parameters revealed any signiÞcant difference between control animals and lead-treated ones. These results are in concordance with previous Þndings of this and similar lead dosing models (18Ð20). In pups, although a signiÞcant reduction in body weight was found since postnatal day 6, no differences were detected in hemoglobin and plasma glucose levels, so it seems that the lead treated pups have a good physiological status. The decrease in the values of hematocrit conÞrm the observation that lead exposure can produce hematological disturbances, including poikilocytosis, perhaps due to the oxidative stress induced by lead in red blood cells (5). Since the blood/brain barrier does not form a substantial impediment to lead entry into the developing brain, the brain lead levels (mg/100g) are about 1Ð3 times the concentration in whole blood (mg/100 ml) (21). Although we had not measured the lead concentration in the brain, we expect neurochemical changes due to lead exposure. We have measured alkaline phosphatase activity (ALP), which is a zinc metalloenzyme involved in both maturation and myelination. The decrease of this enzyme activity could be related to the decrease of zinc concentration described by several authors in lead intoxication (22). Recent studies have suggested that ALP might have more global biological functions because ALP from diverse sources has been shown to have pyrophosphatase activity and
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phosphate ester hydrolysis in biological systems has become recognized as an important process linking energy metabolism and cell signal transduction pathways (23). We also conÞrmed that Pb may exert an inhibitory effect directly on Na1/K1ATP-ase activity in cerebellum and cerebral cortex of intoxicated pups. It is known that Na1/K1ATPase, a key enzyme for maintaining the ion distribution about the cellular membrane which is required for neuronal activity, is among the enzymes particularly affected by lead (24,25). This fact may relate to the different alpha subunit composition of Na1/K1ATP-ase of different tissues. Enzyme extracted from brain has mostly a2 an a3 isozymes present in signiÞcant amounts (26) All the subunits of the enzyme (a1, 2 and 3) differ with the number of reactive sulphydryl groups in the catalytic site (27) and a2 and 3 forms of the enzyme are much more sensitive to the inhibitory effect of Pb than the a1 form is (24). Thus it can be suggested that one of the possible mechanisms of the inhibition may be the interaction of Pb with -SH groups in the catalytic site of the enzyme. The general decrease in neurotransmitters observed in our study could be related with a decreased synthetic capacity, because it has been described that lead could inhibit tyroxine hydroxylase activity (28) or alternatively with lead interference with cellular energy metabolism, inhibiting ATP synthesis that leads to the dysfunction of energy-dependent neuronal events such as neurotransmitter uptake (6). There was not an homogeneous response within the different brain areas. This agrees with others studies where it has been observed that lead, depending on the dose has disparate effects on the neurotransmitter levels in a given region of the brain and that the concentrations of neurotransmitters in various regions of the brain responded differently to the same dose of Pb21. In this sense, previous studies performed in our laboratory, with similar doses of lead acetate, showed normal 5-HT values in presence of increased 5-HIAA on postnatal day 0 and reduced 5-HT with reduced 5-HIAA values on postnatal day 5 (20). Likewise, it has been described (18) in a pre- and postnatal lead exposure that produced blood levels of 50Ð60 mg/100 mL, a signiÞcant reduction of 5-HT in brain stem, whereas 5-HIAA was reduced both in the brain stem and striatum. However, when lead was administrated in adult rats, although the blood levels reached were similar, only the hypothalamus showed a signiÞcant increase in DOPAC levels, whereas the rest of brain areas studied (striatum, hippocampus, midbrain and cortex) did not show changes in monoamine levels (29). This could not be related with a preferential accumulation of lead, since it has been described a homogeneous distribution of lead within the brain (19), but with local differences in the formation and maturation of the different brain regions and transmitter identiÞed systems. Monoamine tissue levels usually provide a good estimate of the innervation density (30), and these results indicate that the monoamine Þbers (i.e. the synaptogenesis) were affected by the gestational and lactational lead exposure and it could be reßected as later behavioral changes. In this sense, the hippocampus was chosen as the anatomical site for learning and memory and as an important target of neurotoxic agents. The neuroanatomical and functional development of the rat hippocampus extend up to the second postnatal week. The experimental paradigm of lead exposure was designated to include this period. In fact, this area was the most affected in relation to the decrease in neurotransmitters as well as the diminution of energy charge. This fact can be related with the impairment of long-term potentiation and learning following perinatal lead exposure described in the literature (31). Likewise, the disturbed
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striatal and cerebellar functions may contribute to the spectrum of symptoms such as hyperactivity and motor coordination problems associated with lead exposure (1). In our work, the chronic pre- and postnatal exposure of rats to low levels of lead via dams resulted in blood concentrations in the offspring of about 29 mg/dL, a value only slightly higher than levels found in children environmentally exposed to this pollutant. This in turn points to the need to deÞne and follow behavioral and neurophysiological biomarkers of lowlevel lead exposure. References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31.
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