Prenatal malnutrition and lead intake produce increased brain lipid peroxidation levels in newborn rats
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Brenda Gabriela Maldonado-Cedillo 1, Araceli Díaz-Ruiz 2, Sergio Montes2, Sonia Galván-Arzate2, Camilo Ríos2,3, Vicente Beltrán-Campos 4, Mireya AlcarazZubeldia 2, Sofia Díaz-Cintra1 1
Departamento de Neurofisiología del Desarrollo y Neurofisiología, Instituto de Neurobiología Campus UNAMJuriquilla, Santiago de Querétaro, Querétaro, Mexico, 2Departamento de Neuroquímica, Instituto Nacional de Neurología y Neurocirugía, Ciudad de México, DF, México, 3Departamento de Sistemas Biológicos de la Universidad Autónoma Metropolitana, Unidad Xochimilco México, Delegación Coyoacán, DF, México, 4División de Ciencias de las Salud e Ingenierías, Universidad de Guanajuato, Campus Celaya-Salvatierra, Leon, GTO, Mexico
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Objectives: Prenatal malnutrition (M) and lead intoxication (Pb) have adverse effects on neuronal development; one of the cellular mechanisms involved is a disruption of the pro- and anti-oxidant balance. In the developing brain, the vulnerability of neuronal membrane phospholipids is variable across the different brain areas is variable. This study assesses the susceptibility of different brain regions to damage by tissue oxidative stress and lead concentrations to determine whether the combined effect of prenatal malnutrition (M) and lead (Pb) intoxication is worse than the effect of either of them individually. Methods: M was induced with an isocaloric and hypoproteinic (6% casein) diet 4 weeks before pregnancy. Intoxication was produced with lead acetate in drinking water, from the first gestational day. Both the M and Pb models were continued until the day of birth. Four brain regions (hippocampus, cortex, striatum, and cerebellum) were dissected out to analyze the lipid peroxidation (LP) levels in four groups: normally nourished (C); normally nourished but intoxicated with lead (CPb); malnourished (M); and M intoxicated with lead (MPb). Results: Dam body and brain weights were significantly reduced in the fourth gestational week in the MPb group. Their pups had significantly lower body weights than those in the C and CPb groups. The PbM group exhibited significant increases of lead concentration and LP in all areas evaluated. A potentiation effect of Pb and M on LP was found in the cerebellum. Discussion: This study provides information on how environmental conditions (intoxication and malnutrition) during the intrauterine period could differentially affect the development of neuronal plasticity and, in consequence, alter adult brain functions such as learning and memory.
Introduction
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Keywords: Lead, Prenatal malnutrition, Lipid peroxidation, Neonatal brain, Hypoproteinic diet
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Nutrition plays an important role in developmental, biochemical, genetic, and molecular mechanisms.1 Owing to protein deficiency, malnutrition during gestation and development can cause structural and biochemical abnormalities in different brain regions2,3 such as the neocortex4,5 and the hippocampus, including dendrite abnormalities6–8 and alterations in GABAergic neurons.9,10 Malnutrition over long periods of time produced maladaptive emotional and Correspondence to: Sofía Díaz-Cintra, Instituto de Neurobiología, Campus UNAM Juriquilla, Boulevard Juriquilla 3001, Querétaro 76230, México. Email: [email protected]
© W. S. Maney & Son Ltd 2015 DOI 10.1179/1476830515Y.0000000003
motivational behaviors,11 changes in anxiety-related behavior and cardiovascular parameters,12 and deficits in learning and memory.13,14 Lead (Pb) also is a neurotoxic agent in the environment, and its effects are manifested in the developing brain,15 producing delays in its maturation,16 and neuronal changes that may compromise adult cognitive, emotional, and motor functions by noradrenaline depletion.17 In children, there is a correlation between Pb exposure and neurological dysfunction.18 Exposure to Pb in early life has been implicated in subsequent progression of amyloidogenesis in rodents during aging.19 Pb can have an impact on brain processes
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Prenatal malnutrition and lead intake produce brain LP levels in newborn rats
throughout the experiment. Animals were maintained with a 12-h/12-h inverted light/dark cycle (lights off at 9:00 h) at 22–23°C and 40–50% humidity.
Experimental procedure Rats were separated into four groups, 10 animals per group: (i) control (C) group of well-nourished animals that were fed a standard rodent diet (Purina 5001 Chow with 23.4% casein); (ii) well-nourished but intoxicated with Pb (CPb); (iii) malnourished groups (M) in which malnutrition was induced with an isocaloric and hypoproteinic (6% casein) diet (Harlan Teklad diet, Madison, WI, USA; TD 92090) starting 4 weeks before mating and continued throughout gestation (21 days), and (iv) M group with the same isocaloric and hypoproteinic, 6% casein diet, but also intoxicated with Pb (MPb). For mating, one male was placed in a cage containing two females, and vaginal smears were obtained each morning to determine the onset of pregnancy, as indicated by the presence of sperm in the smear. To start lead intoxication in rats from groups CPb and MPb, the lead acetate [(CH3COO)2 Pb, Mallinckrodt Baker (JT), México)] was added to the drinking water (concentration of 160 ppm). Daily water and food intake of dams were recorded. Body weights of the 40 pregnant rats were registered weekly starting 1 week before mating until the delivery day (data for a total of 4 weeks, Fig 1). From the litters of the four groups (C, CPb, M, and MPb), six newborn from both sexes per litter were randomly selected for sacrifice, on post-natal day 0 (P0) giving a total of 24 pups. Before decapitation, the body weight of every pup was registered. We previously reported that LP is not influenced by the sex of the pups.31 The brain dissections were made on ice, and immediately after opening the skull, the cerebellum was removed, followed by the separation of the cortex, hippocampus, and striatum with the aid of a stereoscopic microscope (Leica S6D). The tissues were collected in Eppendorf tubes, frozen immediately, and stored at −75°C until use for LP analysis. In addition, all dams (40) were perfused transcardially with a buffered solution of 10% formalin ( pH 7.4), and their brains were removed and weighed.
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Methods and material
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(synthesis, storage, release of neurotransmitters, and receptor function) and cause glial damage.20 The toxicity of Pb is often attributed to its ability to interfere with cellular calcium-dependent processes due to its chemical similarity to calcium ions.21 In animal models of Pb neurotoxicity, deficits in synaptic plasticity and in learning and memory have been observed.20 Protein malnutrition as well as exposure to Pb prior to weaning has an impact on the hippocampal redox state.22–24 For example, increased lipid peroxidation (LP) levels in the hippocampus of malnourished offspring22 and catalase CAT down-regulation during pre- and neonatal Pb exposure24 have been reported. The brain is considered particularly vulnerable to oxidative damage caused by Pb intoxication which contributes to tissue injury and down-regulation of several endogenous antioxidant enzymes and peptides such as superoxide dismutase, CAT, and glutathione.25,26 Phospholipids are important constituents of neuronal membranes and are among the molecules most affected by LP, which results in several pathological conditions.27–30 Malnutrition and Pb are two risk factors during the development of the nervous system, though the cellular mechanisms of their action are not fully understood. Here, we propose that malnutrition and lead (as a neurotoxic agent) administered during pregnancy could have a strong effect on vulnerable brain areas such as the hippocampus and the neocortex, which could be reflected in functional alterations in adult life. Furthermore, measuring LP can answer the question of whether Pb exposure is equally detrimental for different brain areas during development or is worse if combined with malnutrition. Thus, the aim of our study was to explore if prenatal malnutrition and Pb intoxication, or the combination of these two stressors during gestation have differential effects on Pb concentrations and the level of LP (as an indicator of oxidative stress) in four different vulnerable brain regions (hippocampus, frontal cortex, striatum, and cerebellum) of newborn rats.
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A total of 40 Sprague-Dawley female rats, 70–90 days of age (270–290 g), were used and treated in accordance with the NIH Guide for Care and Use of Laboratory Animals, and the local Animal Care and Use Committee approved the experiments. Animal care and management were supervised by a licensed veterinarian, in accordance with the Bioethics Committee of the Instituto de Neurobiología – Universidad Nacional Autónoma de México INBUNAM. Rats were housed in polycarbonate cages (45 × 24 × 21 cm) with adequate, clean nesting material, and free access to food and purified water
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Analysis of lead concentration in tissue Brain tissue was carefully weighed into propylene tubes, and then 200 μl of Suprapur® nitric acid (Merck, Darmstadt, Germany) was added; samples were then capped and placed in a water bath at 45°C for 30 min to achieve digestion. Lead was assayed by graphite furnace atomic absorption using the Zeeman background correction (Spectrophotometer Perkin-Elmer model AA600, autosampler AS-800). Calibration curves were constructed with a
Prenatal malnutrition and lead intake produce brain LP levels in newborn rats
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Figure 1 Dam’s body weights recorded weekly, starting 1 week before mating (Week 1) and then, once a week during pregnancy (Weeks 2, 3 and 4). C: rats fed with the standard diet without lead acetate (control group), CPb: animals fed with the standard diet and intoxicated with lead acetate through drinking water, M: malnourished animals without lead, MPb: malnourished rats, exposed to lead acetate through drinking water. The results are expressed as means ± SEM of 10 animals per group. Repeated measures ANOVA tests followed by Fisher’s test. Significant differences F(3,9) = 128.28, *P < 0.001, and interactions between two variables F(9,108) = 13.70 P < 0.0001 were found between the MPb and the other groups at Week 4.
protein concentration of a one-milliliter aliquot collected from the same homogenate used for LFP was estimated according to the Lowry assay, using bovine serum albumin as a standard.35 We choose LFP as a marker of LP because it is closely correlated with lead-induced brain damage.27
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commercial standard (GFAAS mixed standard, Perkin-Elmer Pure). Matrix modifier consisting of Triton X-100 (0.05%) and monobasic ammonium phosphate (0.01%) was used to dilute standards and samples. Samples were assayed in duplicate, and standard deviations were less than 10% between repetitions. Results are expressed as microgram of lead per gram of wet tissue.32
Lipid peroxidation assay
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Lipid peroxidation was estimated, as described by us previously.27 In brief, each brain region was homogenized in 3 ml of saline solution (0.9% NaCl in distilled water). One-milliliter aliquots of the homogenate were then transferred into capped, glass tubes, and 3 ml of a chloroform–methanol 2:1 (v/v) solution was added. Tubes were gently mixed for 2 min and then placed on ice for 30 min to allow phase separation. The aqueous phase was discarded; 0.9 ml of the chloroform layer was transferred into a quartz cuvette and 0.1 ml of methanol was added. Fluorescence was measured in a Perkin-Elmer LS50B luminescence spectrophotometer (excitation 370 nm, emission 430 nm). Before the sample measurements, the fluorimeter was adjusted to 150 fluorescence units with a 0.1 mg/l quinine standard, prepared in a 0.05 M aqueous sulfuric acid solution. Results were expressed as fluorescence units per milligram of protein (FU/mg of protein). Samples from each brain region were measured in duplicate. The formation of lipid fluorescent products (LFP), as an index of LP, was monitored in the selected brain regions using the technique described by Triggs and Willmore33 and modified by Santamaria et al. 34 The
Statistical analysis Significant differences in Longa scores (dam weights and their water and food intakes) were determined using the repeated-measures ANOVA followed by Fisher’s protected least significance difference post hoc test (PLSD). Also, for multiple comparisons between groups two-way ANOVA tests for dam and body weights of newborn rats followed by Fisher’s test were made. The results of lead concentration in tissue and LP were analyzed with the Kruskal–Wallis test followed by Mann–Whitney U test, as data showed no variance homogeneity (Levene’s test for homogeneity of variances). All analyses were performed by using the SPSS 19.0 software. Values with P < 0.05 were considered significant.
Results Dam’s daily water and food intake The average water intake in milliliter ( per dam per day) was for C, 41.0 ml; for CPb, 37.40 ml groups and 36.69 ml; for M; and for MPb 37.48 groups; differences across the four groups [F(3,36) = 2.26, P = 0.1007] were not significant. The ANOVA revealed significant differences in food intake among groups [F(3,36) = 5.789, P = 0.0025], and the post hoc PLSD test showed significant (P < 0.01) reductions between the C vs. M groups.
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Prenatal malnutrition and lead intake produce brain LP levels in newborn rats
Dam’s body and brain weights The ANOVA of repeated measures (across 4 weeks) revealed statistically significant differences F(3,36) = 3.85, P < 0.05 in the dam’s body weights among groups, and the post hoc PLSD test showed that weights of dams of the MPb group were significantly (P < 0.01) lower than those of the, C, CPb, and M groups in the fourth gestational week. Significant differences F(3,9) = 128.28, P < 0.001, and interactions among the four groups F(9,108) = 13.70, P < 0.0001, were also found as shown in Fig. 1. The comparison of mother brain weights revealed significant differences F(3,36) = 3.837, P = 0.0176 among groups, and the post hoc PLSD test showed significantly lower brain weights in the MPb vs. C (P < 0.0076) and in the MPb vs. M (P < 0.0044) comparisons (Fig. 2).
Body weights of newborn rats
60.3 ± 20.5
(P < 0.05,
Brain cortex Lead concentration in the cerebral cortex was significantly different among groups (Kruskal–Wallis test, P = 0.0001). Lead concentration in the cerebral cortex was increased in both of the two groups of rats exposed to lead (37.7 ± 9.2 and 35.4 ± 5.7, respectively). These values were significantly different (P < 0.05) from those of the malnourished animals (M) 12.9 ± 2.6. Finally, the C group had lead levels of 23.6 ± 3.6 (Table 1).
Striatum The average values observed in the striatum of newborn animals (P0) is shown in Table 1. As we can see, there is a statistically significant increase (Kruskal–Wallis test P = 0.006 followed Mann–Whitney U test, P < 0.05) in lead concentrations in the CPb (43.3 ± 4.9) and in the MPb (38.5 ± 14.9) group when compared with the groups well-nourished and lead-free (C) and lead intoxicated (Pb) groups (18.4 ± 4.0 and 13.5 ± 3.5, respectively).
Cerebellum Lead intoxication in the hippocampus of newborn rats is shown in Table 1, and was significantly different among groups after Kruskal–Wallis tests, (P = 0.007); as shown, there are average lead levels of 25.0 ± 3.5 in C animals (without malnutrition and lead intoxication), whereas results obtained in M (malnourished and free-of-lead) animals were of 17.5 ± 5.0. In animals well-nourished and intoxicated with lead, the value was 53.6 ± 8.5 that is statistically significant compared to C and M groups (P < 0.05). Finally, the lead average value of the malnourished
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The two-way ANOVA revealed significant differences F(3,20) = 49.10, P < 0.0001 in the body weights among groups, and the post hoc PLSD test showed significantly lower (P < 0.001) body weights for the M and MPb groups of newborn rats when compared with the C and Pb groups. Moreover, the MPb group showed a significantly lower (P < 0.001) body weight than the M group (Fig. 3).
average value was Mann–Whitney U test).
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Lead concentrations in newborn-rat brains
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Hippocampus The lead concentration in the hippocampus of newborn rats were significantly different among groups, after the Kruskal–Wallis test, (P = 0.029) (Table 1). Likewise, the observed mean lead levels in C animals were 28.0 ± 6.3, while Pb animals averaged 62.5 ± 9.5 (P < 0.05). M animals showed an average value of 32.1 ± 9.9, while in MPb animals the
Figure 2 Dam’s brain weights measured immediately after delivery. C: animals under the normal diet without lead intoxication; Pb: rats under the normal diet and intoxicated with lead; M: malnourished rats without lead intoxication; and MPb: malnourished and lead-intoxicated rats. The results are expressed as means ± SEM of 10 animals per group. Oneway ANOVA followed by Fisher’s post hoc test. *P < 0.05 vs. C and M groups
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Figure 3 Newborn body weights measured immediately after birth. C: animals under normal diet without lead intoxication; Pb: rats under normal diet and intoxicated with lead; M: malnourished rats without lead intoxication; and MPb: malnourished and lead-intoxicated rats. The results are expressed as means ± SEM of six randomly selected animals per group. Two-way ANOVA followed by Fisher’s post hoc test. *P < 0.05 CPb vs. C, **P < 0.05 M different of CPb and C and †P < 0.05 MPb different from all groups.
12.3
17.9
13.5
17.5
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Effects of lead intoxication and malnutrition on LP levels
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31.0 48.1 53.6*
25.0 45.1 43.3** 20.4 16.1 18.4 Striatum
and lead intoxicated group was 51.5 ± 12.3, with significant differences when compared with C and M groups.
Hippocampus LP levels in the hippocampus of newborn rats are shown in Table 2. The results are expressed as fluorescence units per milligram of protein, the basal LP was of 23.5 ± 2.5 (control, group), likewise for the malnutrition (M) group the results are 49.3 ± 14.5. Analysis of hippocampal tissue revealed a significant increase of LP (P < 0.05) in the M and MPb groups when compared with the C and CPb groups. Lead intoxication effects were not found to differ significantly between the C and CPb or between the M and MPb groups.
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9.4 12.9 15.2 27.3 37.7* 19.1 21.9
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Prenatal malnutrition and lead intake produce brain LP levels in newborn rats
Brain cortex Cerebral cortex tissue showed an increase (P < 0.05) of LP in both M and MPb when compared with the C group (35.9 ± 3.0). There was neither an additive nor a synergistic effect of M and Pb, because there was no difference between malnourished groups (M vs. MPb). A similar difference was observed between the C and CPb groups in animals only exposed to Pb with normal diet, CPb (Table 2).
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22.4
21.1
13.0
26.3 32.1
23.1 108.5 13.8 119.7 13.4 61.0 23.7 114.3 52.3 60.5 62.5**
4.7 47.7 12.5 42.0 6.3 37.8 9.3 43.0 33.9 28.0 Hippocampus
28.6
Results are expressed in ng/g of wet tissue. The Kruskal–Wallis test followed by the Mann–Whitney U test. IQR, interquartile range; C, animals under normal diet and without lead; Pb, rats fed normal diet and exposed to lead; M, malnourished rats without lead exposure; and MPb, malnourished rats exposed to lead. Significant differences **P < 0.05 compared to C and M groups and *P < 0.05 compared to the M group.
70.4 55.5 51.5*
41.3 12.7 38.5**
35.4*
30.7
22.3
4.4 286.7 4.4 88.0 2.1 185.4 12.8 100.0 42.2 38.6 60.3**
1.8 148.3 3.6 46.2 0.5 45.8 0.2 53.6 27.7
Median IQR Median IQR Median Mean
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Table 1 Lead concentrations in newborn-rat brains
Min Max
Mean
CPb
Min Max
Mean
Median
M
IQR
Min Max
Mean
MPb
IQR
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Striatum Similar effects to those found in the cortex were observed in the striatum tissue; both the M and the MPb groups showed a significant increase (P < 0.05) of LP when compared with the C and CPb groups (Table 2). Cerebellum LP in the cerebellum showed a statistically significant increase (P < 0.05) with M both alone and in combination with lead (MPb), when compared with the C group (60.5 ± 3.2) (Kruskal–Wallis test followed by Mann–Whitneys U test). In addition, the post hoc Mann–Whitney U test showed no differences in LP levels in C vs. CPb (Table 2). Interestingly, for this region, a significant difference was found between the group M and group M/Pb (P < 0.05), indicating a significant interaction between Pb and M.
Discussion Our findings demonstrated heterogeneous effects on the concentrations of both LP and Pb measurements in four neonatal brain areas in response to protein restriction and lead intoxication during the gestational period. Both agents produce a significant reduction of body weights and differential oxidative damage in the brain, but the significant increase of the Pb concentrations and LP is more evident in the four newborn
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60.6 60.5 Cerebellum
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85.1 87.2* 93.2 57.7 71.0
30.7 43.9 45.2
51.7 40.4 47.7 11.8 34.5
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22.8 23.5 Hippocampus
Median Mean
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brain regions analyzed. During gestation the dams water intake was similar in the four groups (C, CPb, M, and MPb); however, a significant reduction in food intake was found in the M and MPb groups; in this group we also found a reduction in the body weight. It has been reported that chow consumption is different in the group of rats under low-protein diet, as a result of endocrine signals.36 Malnutrition (M) during the pre- and postnatal periods affects a large number of children worldwide, resulting in biochemical, physiological, and anatomical changes in the central nervous system that are associated with cognitive and behavioral impairment during childhood and adolescence.36 In animals, the protein content of the diet plays an important role in antioxidant mechanisms in the brain, and malnutrition produces an overproduction of reactive oxygen species (ROS) that lead to membrane oxidation and thus to an increase in LP due to changes in oxidant and antioxidant levels in the plasma.37 Protein malnutrition may lead to an increase in oxidative damage by diminishing antioxidant defenses of the brain.26,27 On the other hand, intrauterine exposure to a mixture of environmental pollutants has become a major health concern, because it could adversely affect the development trajectory during pregnancy.38 Lead exerts its most severe effects on the developing brain, which can be attributed to the intense cell proliferation and synaptogenesis which takes place in the various more sensitive brain areas such as the hippocampus, frontal cortex, cerebellum, and striatum.15 In fact, those areas also accumulated more lead, among the various brain regions examined (article by Villeda-Hernandez et al.). Lead differential distribution among brain regions may reflect, in turn, vascularization of the different brain regions. Furthermore, one of the important mechanisms of action of environmental chemicals may involve ROSinduced oxidative stress. 39 In the brain, chronic exposure to lead would predominantly affect two specific protein complexes: protein kinase C and the N-methyl-D-aspartate (NMDAR) subtype of the glutamate receptor. There is a hypothesis that NMDAR-receptor-dependent and protein synthesisdependent long-term potentiation are important targets for neurotoxicity induced by Pb.40 Both factors, Pb intoxication and M with only a 7% of casein, during gestation, produced increased oxidative damage to lipids and proteins.29,30 Therefore, it is necessary to understand the consequences of these adverse effects during brain development and the resulting functional impairments such as memory disturbance, learning deficits, and behavioral problems. Populations with limited economic resources may also have nutritional deficits that make them uniquely
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16.4
49.8 91.0 94.3**
57.6 57.7*
92.5 97.2**
20.3 102.6 16.8 87.3 27.2 64.9 17.9 173.1 67.1 33.5 49.3
20.2 34.8 28.5 47.3 34.8 67.0 50.2 72.5 11.3
Median IQR
Min Max
Results are expressed in FU/mg of protein. The Kruskal–Wallis test followed by the Mann–Whitney U test. IQR, interquartile range; C, animals under normal diet without lead intoxication; Pb, rats under normal diet and intoxicated with lead; M, malnourished rats without lead intoxication; and MPb, malnourished and lead-intoxicated rats. Significant differences, *P < 0.05 vs. C, **P < 0.05 vs. C and CPb and †P < 0.05 vs. all other groups.
126.4 171.0 161.4†
54.9 104.5 118.2**
35.7 86.4 78.6*
22.4 84.5 84.3**
71.5 139.3 34.4 76.2 63.3 145.9 65.8 111.4 18.2
IQR 6
C
Table 2 Lipid peroxidation levels in newborn-rat brains
Mean
CPb
IQR
Min Max
Mean
Median
M
46.2
IQR Median Mean
MPb
Min Max
67.2 97.4 51.3 97.2 75.3 228.8 78.9 253.1
Prenatal malnutrition and lead intake produce brain LP levels in newborn rats
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establishment of oxidative stress, mainly in the cerebellum, indicating its higher vulnerability to lead toxicity in newborn rats. Similar effects have been described by Mousa et al.,46 who reported increase of fetal mortality and reduced body growth and brain weight due to lead intoxication; they also found fetal cerebellar damage in cortical layers, degenerative changes including formation of clumps of Nissl granules in Purkinje and granule cells, and finally, an increase neuronal apoptosis. Further experiments are needed to explain how malnutrition and lead intoxication lead to the diminished adult brain weights; we observed since another reports show diminished brain/body weights, only after 1 month of lead exposure (100 mg/kg/day).47,48 Loss of tissue, both in the adult and developing brain as a result of Pb exposure and malnutrition, may be the result of enhanced brain cells’ death, which in turn may be reflected as a reduced brain weight. This explanation remains speculative until further experiments test that hypothesis. Likewise, pre- and postnatal level of the 4-hyrdoxynanoneal (a byproduct of LP), and the effect of oxidative stress due to combined exposure to malnutrition and Pb remains a central subject to be examined in future experiments. Studies should include the response of antioxidant enzymes, such as catalase and/or superoxide dismutase, which have been reported to be increased during the postnatal period,15 in order to determine whether the effects of oxidative stress correspond to the time of neuronal differentiation in each brain region, and if they correlate with age-related changes in the capacity of the antioxidant system.
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susceptible to Pb intoxication.18,41 Pb is naturally present in nature; however, most of the high levels found in the environment come from human activities. Lead-induced enhancement of LP and redox imbalance in the brain are possible mechanisms responsible for brain damage. 27 The central nervous system is most sensitive to Pb exposure during early development due to rapid cell proliferation and migration, axonal growth, synaptogenesis, and neurotransmitter systems such as the GABAergic interneurons.42 In addition, exposure of animals to Pb has disrupted hippocampal development, with deficits in learning and memory capacities.43 We used a low dose of Pb during the gestational period when the immature rat brain is highly vulnerable. In a similar study, Baranowska-Bosiacka et al. 24 found that rats were highly vulnerable during the perinatal period to Pb exposure at a 10 μg/dl concentration, which resulted in damage to the adult hippocampus. They concluded that oxidative stress is one of the possible mechanisms disturbing cellular metabolism in this structure. During intrauterine malnutrition, female offspring present an increased superoxide production, maybe catalyzed by nitric oxide synthase, whose activity in the hippocampus, the cerebral cortex, and the cerebellum is inhibited by lead exposure to an extent that depends on its concentration and the duration of exposure.44 The lead concentration (ng/g of wet tissue) in C and MPb groups increased significantly in the four areas analyzed (Table 1), suggesting brain hypomyelination due to Pb intoxication and M during pregnancy. These concomitant effects were observed in the brain and the cerebellum in rats of 30, 60, and 120 days of age when Pb was administered on postnatal days 1–15 (P1–P15) by daily intraperitoneal injections of 10 mg lead nitrate/kg body weight.45 Malnutrition resulted in a three-fold higher LP rate in the hippocampus in the M vs. the C group. Nevertheless, the combination of both lead intoxication and M (Table 2) showed only a two-fold increase in LP compared with C, indicating an interaction between the two treatments. Previous malnutrition reports from Feoli et al.29,30 employing a 7% casein diet, demonstrated the damaging consequences within the cerebellum and the cerebral cortex of oxidative stress, especially damage of macromolecules and the increased levels of LP. These authors also reported a significant decrease in total antioxidant levels in the cerebral cortex of protein-malnourished rats. Our data indicated that prenatal protein malnutrition with a 6% casein diet during pregnancy increased the oxidative damage to brain lipids. On the other hand, when this treatment (water containing 160 ppm lead) was administered to animals exposed to the malnutrition model, the results showed the
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Acknowledgements This study was supported by DGAPA-UNAM and CONACYT. The authors wish to thank A. Aguilar and M. García for technical assistance. We are grateful to M. Salas and S. Castro-Chavira for their invaluable suggestions on the manuscript and to D. Pless for proofreading.
Disclaimer statements Contributors None. Funding None. Conflicts of interest None. Ethics approval None.
References 1 Stover PJ, Garza C. Nutrition and developmental biology— implications for public health. Nutr Rev 2006;64:S60–71; discussion S72–91. 2 Morgane PJ, et al. Prenatal malnutrition and development of the brain. Neurosci Biobehav Rev 1993;17:91–128.
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25 Adonaylo VN, Oteiza PI. Pb2+ promotes lipid oxidation and alterations in membrane physical properties. Toxicology 1999; 132:19–32. 26 Hsu P-C, Guo YL. Antioxidant nutrients and lead toxicity. Toxicology 2002;180:33–44. 27 Villeda-Hernández J, et al. Morphometric analysis of brain lesions in rat fetuses prenatally exposed to low-level lead acetate: correlation with lipid peroxidation. Histol Histopathol 2006;21:609–17. 28 Reitz C, Brayne C, Mayeux R. Epidemiology of Alzheimer disease. Nat Rev Neurol 2011;7:137–52. 29 Feoli AM, et al. Brain glutathione content and glutamate uptake are reduced in rats exposed to pre- and postnatal protein malnutrition. J Nutr 2006;136:2357–61. 30 Feoli AM, et al. Effects of protein malnutrition on oxidative status in rat brain. Nutrition 2006;22:160–5. 31 Gutiérrez-Reyes EY, Albores A, Ríos C. Increase of striatal dopamine release by cadmium in nursing rats and its prevention by dexamethasone-induced metallothionein. Toxicology 1998; 131(2–3):145–54. 32 Montes M, Iturriza-Gómara M. Molecular methods for the diagnosis of acute viral gastroenteritis. Enferm Infec Microbiol Clin 2008;26(Suppl. 9):81–5. 33 Triggs WJ, Willmore LJ. In vivo lipid peroxidation in rat brain following intracortical Fe2+ injection. J Neurochem 1984;42: 976–80. 34 Santamaría A, Ríos C. MK-801, an N-methyl-D-aspartate receptor antagonist, blocks quinolinic acid-induced lipid peroxidation in rat corpus striatum. Neurosci Lett 1993;159:51–4. 35 Lowry O, Rosebrough ., Farr AL, Randall RJ. Protein measurement with the Folin phenol reagent. J Biol Chem 1951;193:265–75. 36 Laeger T, et al. FGF21 is an endocrine signal of protein restriction. J Clin Invest. 2014;124(9):3913–22. 37 Khare M, Mohanty C, Das BK, Jyoti A, Mukhopadhyay B, Mishra SP. Free radicals and antioxidant status in protein energy malnutrition. Int J Pediatr 2014;254396. 38 Rider CV, Furr JR, Wilson VS, Gray LE. Cumulative effects of in utero administration of mixtures of reproductive toxicants that disrupt common target tissues via diverse mechanisms of toxicity. Int J Androl 2010;33:443–62. 39 Luo ZC, Liu JM, Fraser WD. Large prospective birth cohort studies on environmental contaminants and child health-Goals, challenges, limitations and needs. Med Hypotheses 2010;74: 318–32. 40 Petit TL, LeBoutillier JC, Brooks WJ. Altered sensitivity to NMDA following developmental lead exposure in rats. Physiol Behav 1992;52:687–93. 41 Ferlemi A-V, et al. Lead-induced effects on learning/memory and fear/anxiety are correlated with disturbances in specific cholinesterase isoform activity and redox imbalance in adult brain. Physiol Behav 2014;131C:115–122. 42 Wirbisky SE, Weber GJ, Lee JW, Cannon JR, Freeman JL. Novel dose dependent alterations in excitatory GABA during embryonic development associated with lead (Pb) neurotoxicity. Toxicol Lett 2014;229(1):1–8. 43 Moreira EG, Vassilieff I, Vassilieff VS. Developmental lead exposure: behavioral alterations in the short and long term. Neurotoxicol Teratol 2001;23:489–95. 44 Zhu Z, Yang R, Dong G, Zhao Z. Study on the neurotoxic effects of low-level lead exposure in rats. J Zhejiang Univ Sci B 2005;6:686–92. 45 Sundström R, Karlsson B. Myelin basic protein in brains of rats with low dose lead encephalopathy. Arch Toxicol 1987;59(5):341–5. 46 Mousa AM, Al-Fadhli AS, Rao MS, Kilarkaje N. Gestational lead exposure induces developmental abnormalities and up-regulates apoptosis of fetal cerebellar cells in rats. Drug Chem Toxicol 2014; Apr 13 (Epub ahead of print). 47 Khalaf AA, Moselhy WA, Abdel-Hamed MI. The protective effect of green tea extract on lead induced oxidative and DNA damage on rat brain. Neurotoxicology 2012;33(3):280–9. 48 Galler JR, et al. Malnutrition in the first year of life and personality at age 40. J Child Psychol Psychiatry 2013;54(8):911–9.
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3 Léonhardt M, et al. Perinatal maternal food restriction induces alterations in hypothalamo-pituitary-adrenal axis activity and in plasma corticosterone-binding globulin capacity of weaning rat pups. Neuroendocrinology 2002;75:45–54. 4 Cordero ME, Valenzuela CY, Rodriguez A, Aboitiz F. Dendritic morphology and orientation of pyramidal cells of the neocortex in two groups of early postnatal undernourished-rehabilitated rats. Brain Res Dev Brain Res 2003;142:37–45. 5 Salas M, Torrero C, Rubio L, Regalado M. Effects of perinatal undernutrition on the development of neurons in the rat insular cortex. Nutr Neurosci 2012;15:20–5. 6 García-Ruiz M, Díaz-Cintra S, Cintra L, Corkidi G. Effect of protein malnutrition on CA3 hippocampal pyramidal cells in rats of three ages. Brain Res 1993;625:203–12. 7 Cintra L, et al. Effects of prenatal protein malnutrition on hippocampal CA1 pyramidal cells in rats of four age groups. Hippocampus 1997;7:192–203. 8 Zhang L, Corona-Morales AA, Vega-González A, GarcíaEstrada J, Escobar A. Dietary tryptophan restriction in rats triggers astrocyte cytoskeletal hypertrophy in hippocampus and amygdala. Neurosci Lett 2009;450:242–5. 9 Díaz-Cintra S, et al. Protein malnutrition differentially alters the number of glutamic acid decarboxylase-67 interneurons in dentate gyrus and CA1-3 subfields of the dorsal hippocampus. Exp Neurol 2007;208:47–53. 10 Lister JP, et al. Prenatal protein malnutrition alters the proportion but not numbers of parvalbumin-immunoreactive interneurons in the hippocampus of the adult Sprague-Dawley rat. Nutr Neurosci 2011;14:165–78. 11 Da Silva Hernandes A, Françolin-Silva AL, Valadares CT, Fukuda MTH, Almeida SS. Effects of different malnutrition techniques on the behavior of rats tested in the elevated Tmaze. Behav Brain Res 2005;162:240–5. 12 Watkins AJ, Sinclair KD. Paternal low protein diet affects adult offspring cardiovascular and metabolic function in mice. Am J Physiol Heart Circ Physiol 2014;306:H1444–52. 13 Valadares CT, de Sousa Almeida S. Early protein malnutrition changes learning and memory in spaced but not in condensed trials in the Morris water-maze. Nutr Neurosci 2005;8:39–47. 14 Cardoso A, Castro JP, Pereira PA, Andrade JP. Prolonged protein deprivation, but not food restriction, affects parvalbumin-containing interneurons in the dentate gyrus of adult rats. Brain Res 2013;1522:22–30. 15 Bokara KK, et al. Lead-induced increase in antioxidant enzymes and lipid peroxidation products in developing rat brain. Biometals 2008;21:9–16. 16 Julvez J, Grandjean P. Neurodevelopmental toxicity risks due to occupational exposure to industrial chemicals during pregnancy. Ind Health 2009;47:459–68. 17 Sabbar M, Delaville C, De Deurwaerdère P, Benazzouz A, Lakhdar-Ghazal N. Lead intoxication induces noradrenaline depletion, motor nonmotor disabilities, and changes in the firing pattern of subthalamic nucleus neurons. Neuroscience 2012;210:375–83. 18 Bellinger DC. Neurological and behavioral consequences of childhood lead exposure. PLoS Med 2008;5:e115. 19 White LD, et al. New and evolving concepts in the neurotoxicology of lead. Toxicol Appl Pharmacol 2007;225:1–27. 20 Sansar W, Ahboucha S, Gamrani H. Chronic lead intoxication affects glial and neural systems and induces hypoactivity in adult rat. Acta Histochem 2011;113:601–7. 21 Goldstein GW. Evidence that lead acts as a calcium substitute in second messenger metabolism. Neurotoxicology 14:97–101. 22 Bonatto F, et al. Effect of protein malnutrition on redox state of the hippocampus of rat. Brain Res 2005;1042(1):17–22. 23 Partadiredja G, Worrall S, Bedi KS. Early life undernutrition alters the level of reduced glutathione but not the activity levels of reactive oxygen species enzymes or lipid peroxidation in the mouse forebrain. Brain Res 2009;1285:22–9. 24 Baranowska-Bosiacka I, et al. Disrupted pro- and antioxidative balance as a mechanism of neurotoxicity induced by perinatal exposure to lead. Brain Res 2012;1435:56–71.
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Brenda Gabriela Maldonado-Cedillo 1, Araceli Díaz-Ruiz 2, Sergio Montes2, Sonia Galván-Arzate2, Camilo Ríos2,3, Vicente Beltrán-Campos 4, Mireya AlcarazZubeldia 2, Sofia Díaz-Cintra1 1
Departamento de Neurofisiología del Desarrollo y Neurofisiología, Instituto de Neurobiología Campus UNAMJuriquilla, Santiago de Querétaro, Querétaro, Mexico, 2Departamento de Neuroquímica, Instituto Nacional de Neurología y Neurocirugía, Ciudad de México, DF, México, 3Departamento de Sistemas Biológicos de la Universidad Autónoma Metropolitana, Unidad Xochimilco México, Delegación Coyoacán, DF, México, 4División de Ciencias de las Salud e Ingenierías, Universidad de Guanajuato, Campus Celaya-Salvatierra, Leon, GTO, Mexico
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Objectives: Prenatal malnutrition (M) and lead intoxication (Pb) have adverse effects on neuronal development; one of the cellular mechanisms involved is a disruption of the pro- and anti-oxidant balance. In the developing brain, the vulnerability of neuronal membrane phospholipids is variable across the different brain areas is variable. This study assesses the susceptibility of different brain regions to damage by tissue oxidative stress and lead concentrations to determine whether the combined effect of prenatal malnutrition (M) and lead (Pb) intoxication is worse than the effect of either of them individually. Methods: M was induced with an isocaloric and hypoproteinic (6% casein) diet 4 weeks before pregnancy. Intoxication was produced with lead acetate in drinking water, from the first gestational day. Both the M and Pb models were continued until the day of birth. Four brain regions (hippocampus, cortex, striatum, and cerebellum) were dissected out to analyze the lipid peroxidation (LP) levels in four groups: normally nourished (C); normally nourished but intoxicated with lead (CPb); malnourished (M); and M intoxicated with lead (MPb). Results: Dam body and brain weights were significantly reduced in the fourth gestational week in the MPb group. Their pups had significantly lower body weights than those in the C and CPb groups. The PbM group exhibited significant increases of lead concentration and LP in all areas evaluated. A potentiation effect of Pb and M on LP was found in the cerebellum. Discussion: This study provides information on how environmental conditions (intoxication and malnutrition) during the intrauterine period could differentially affect the development of neuronal plasticity and, in consequence, alter adult brain functions such as learning and memory.
Introduction
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Keywords: Lead, Prenatal malnutrition, Lipid peroxidation, Neonatal brain, Hypoproteinic diet
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Nutrition plays an important role in developmental, biochemical, genetic, and molecular mechanisms.1 Owing to protein deficiency, malnutrition during gestation and development can cause structural and biochemical abnormalities in different brain regions2,3 such as the neocortex4,5 and the hippocampus, including dendrite abnormalities6–8 and alterations in GABAergic neurons.9,10 Malnutrition over long periods of time produced maladaptive emotional and Correspondence to: Sofía Díaz-Cintra, Instituto de Neurobiología, Campus UNAM Juriquilla, Boulevard Juriquilla 3001, Querétaro 76230, México. Email: [email protected]
© W. S. Maney & Son Ltd 2015 DOI 10.1179/1476830515Y.0000000003
motivational behaviors,11 changes in anxiety-related behavior and cardiovascular parameters,12 and deficits in learning and memory.13,14 Lead (Pb) also is a neurotoxic agent in the environment, and its effects are manifested in the developing brain,15 producing delays in its maturation,16 and neuronal changes that may compromise adult cognitive, emotional, and motor functions by noradrenaline depletion.17 In children, there is a correlation between Pb exposure and neurological dysfunction.18 Exposure to Pb in early life has been implicated in subsequent progression of amyloidogenesis in rodents during aging.19 Pb can have an impact on brain processes
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throughout the experiment. Animals were maintained with a 12-h/12-h inverted light/dark cycle (lights off at 9:00 h) at 22–23°C and 40–50% humidity.
Experimental procedure Rats were separated into four groups, 10 animals per group: (i) control (C) group of well-nourished animals that were fed a standard rodent diet (Purina 5001 Chow with 23.4% casein); (ii) well-nourished but intoxicated with Pb (CPb); (iii) malnourished groups (M) in which malnutrition was induced with an isocaloric and hypoproteinic (6% casein) diet (Harlan Teklad diet, Madison, WI, USA; TD 92090) starting 4 weeks before mating and continued throughout gestation (21 days), and (iv) M group with the same isocaloric and hypoproteinic, 6% casein diet, but also intoxicated with Pb (MPb). For mating, one male was placed in a cage containing two females, and vaginal smears were obtained each morning to determine the onset of pregnancy, as indicated by the presence of sperm in the smear. To start lead intoxication in rats from groups CPb and MPb, the lead acetate [(CH3COO)2 Pb, Mallinckrodt Baker (JT), México)] was added to the drinking water (concentration of 160 ppm). Daily water and food intake of dams were recorded. Body weights of the 40 pregnant rats were registered weekly starting 1 week before mating until the delivery day (data for a total of 4 weeks, Fig 1). From the litters of the four groups (C, CPb, M, and MPb), six newborn from both sexes per litter were randomly selected for sacrifice, on post-natal day 0 (P0) giving a total of 24 pups. Before decapitation, the body weight of every pup was registered. We previously reported that LP is not influenced by the sex of the pups.31 The brain dissections were made on ice, and immediately after opening the skull, the cerebellum was removed, followed by the separation of the cortex, hippocampus, and striatum with the aid of a stereoscopic microscope (Leica S6D). The tissues were collected in Eppendorf tubes, frozen immediately, and stored at −75°C until use for LP analysis. In addition, all dams (40) were perfused transcardially with a buffered solution of 10% formalin ( pH 7.4), and their brains were removed and weighed.
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Methods and material
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(synthesis, storage, release of neurotransmitters, and receptor function) and cause glial damage.20 The toxicity of Pb is often attributed to its ability to interfere with cellular calcium-dependent processes due to its chemical similarity to calcium ions.21 In animal models of Pb neurotoxicity, deficits in synaptic plasticity and in learning and memory have been observed.20 Protein malnutrition as well as exposure to Pb prior to weaning has an impact on the hippocampal redox state.22–24 For example, increased lipid peroxidation (LP) levels in the hippocampus of malnourished offspring22 and catalase CAT down-regulation during pre- and neonatal Pb exposure24 have been reported. The brain is considered particularly vulnerable to oxidative damage caused by Pb intoxication which contributes to tissue injury and down-regulation of several endogenous antioxidant enzymes and peptides such as superoxide dismutase, CAT, and glutathione.25,26 Phospholipids are important constituents of neuronal membranes and are among the molecules most affected by LP, which results in several pathological conditions.27–30 Malnutrition and Pb are two risk factors during the development of the nervous system, though the cellular mechanisms of their action are not fully understood. Here, we propose that malnutrition and lead (as a neurotoxic agent) administered during pregnancy could have a strong effect on vulnerable brain areas such as the hippocampus and the neocortex, which could be reflected in functional alterations in adult life. Furthermore, measuring LP can answer the question of whether Pb exposure is equally detrimental for different brain areas during development or is worse if combined with malnutrition. Thus, the aim of our study was to explore if prenatal malnutrition and Pb intoxication, or the combination of these two stressors during gestation have differential effects on Pb concentrations and the level of LP (as an indicator of oxidative stress) in four different vulnerable brain regions (hippocampus, frontal cortex, striatum, and cerebellum) of newborn rats.
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A total of 40 Sprague-Dawley female rats, 70–90 days of age (270–290 g), were used and treated in accordance with the NIH Guide for Care and Use of Laboratory Animals, and the local Animal Care and Use Committee approved the experiments. Animal care and management were supervised by a licensed veterinarian, in accordance with the Bioethics Committee of the Instituto de Neurobiología – Universidad Nacional Autónoma de México INBUNAM. Rats were housed in polycarbonate cages (45 × 24 × 21 cm) with adequate, clean nesting material, and free access to food and purified water
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Analysis of lead concentration in tissue Brain tissue was carefully weighed into propylene tubes, and then 200 μl of Suprapur® nitric acid (Merck, Darmstadt, Germany) was added; samples were then capped and placed in a water bath at 45°C for 30 min to achieve digestion. Lead was assayed by graphite furnace atomic absorption using the Zeeman background correction (Spectrophotometer Perkin-Elmer model AA600, autosampler AS-800). Calibration curves were constructed with a
Prenatal malnutrition and lead intake produce brain LP levels in newborn rats
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Figure 1 Dam’s body weights recorded weekly, starting 1 week before mating (Week 1) and then, once a week during pregnancy (Weeks 2, 3 and 4). C: rats fed with the standard diet without lead acetate (control group), CPb: animals fed with the standard diet and intoxicated with lead acetate through drinking water, M: malnourished animals without lead, MPb: malnourished rats, exposed to lead acetate through drinking water. The results are expressed as means ± SEM of 10 animals per group. Repeated measures ANOVA tests followed by Fisher’s test. Significant differences F(3,9) = 128.28, *P < 0.001, and interactions between two variables F(9,108) = 13.70 P < 0.0001 were found between the MPb and the other groups at Week 4.
protein concentration of a one-milliliter aliquot collected from the same homogenate used for LFP was estimated according to the Lowry assay, using bovine serum albumin as a standard.35 We choose LFP as a marker of LP because it is closely correlated with lead-induced brain damage.27
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commercial standard (GFAAS mixed standard, Perkin-Elmer Pure). Matrix modifier consisting of Triton X-100 (0.05%) and monobasic ammonium phosphate (0.01%) was used to dilute standards and samples. Samples were assayed in duplicate, and standard deviations were less than 10% between repetitions. Results are expressed as microgram of lead per gram of wet tissue.32
Lipid peroxidation assay
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Lipid peroxidation was estimated, as described by us previously.27 In brief, each brain region was homogenized in 3 ml of saline solution (0.9% NaCl in distilled water). One-milliliter aliquots of the homogenate were then transferred into capped, glass tubes, and 3 ml of a chloroform–methanol 2:1 (v/v) solution was added. Tubes were gently mixed for 2 min and then placed on ice for 30 min to allow phase separation. The aqueous phase was discarded; 0.9 ml of the chloroform layer was transferred into a quartz cuvette and 0.1 ml of methanol was added. Fluorescence was measured in a Perkin-Elmer LS50B luminescence spectrophotometer (excitation 370 nm, emission 430 nm). Before the sample measurements, the fluorimeter was adjusted to 150 fluorescence units with a 0.1 mg/l quinine standard, prepared in a 0.05 M aqueous sulfuric acid solution. Results were expressed as fluorescence units per milligram of protein (FU/mg of protein). Samples from each brain region were measured in duplicate. The formation of lipid fluorescent products (LFP), as an index of LP, was monitored in the selected brain regions using the technique described by Triggs and Willmore33 and modified by Santamaria et al. 34 The
Statistical analysis Significant differences in Longa scores (dam weights and their water and food intakes) were determined using the repeated-measures ANOVA followed by Fisher’s protected least significance difference post hoc test (PLSD). Also, for multiple comparisons between groups two-way ANOVA tests for dam and body weights of newborn rats followed by Fisher’s test were made. The results of lead concentration in tissue and LP were analyzed with the Kruskal–Wallis test followed by Mann–Whitney U test, as data showed no variance homogeneity (Levene’s test for homogeneity of variances). All analyses were performed by using the SPSS 19.0 software. Values with P < 0.05 were considered significant.
Results Dam’s daily water and food intake The average water intake in milliliter ( per dam per day) was for C, 41.0 ml; for CPb, 37.40 ml groups and 36.69 ml; for M; and for MPb 37.48 groups; differences across the four groups [F(3,36) = 2.26, P = 0.1007] were not significant. The ANOVA revealed significant differences in food intake among groups [F(3,36) = 5.789, P = 0.0025], and the post hoc PLSD test showed significant (P < 0.01) reductions between the C vs. M groups.
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Dam’s body and brain weights The ANOVA of repeated measures (across 4 weeks) revealed statistically significant differences F(3,36) = 3.85, P < 0.05 in the dam’s body weights among groups, and the post hoc PLSD test showed that weights of dams of the MPb group were significantly (P < 0.01) lower than those of the, C, CPb, and M groups in the fourth gestational week. Significant differences F(3,9) = 128.28, P < 0.001, and interactions among the four groups F(9,108) = 13.70, P < 0.0001, were also found as shown in Fig. 1. The comparison of mother brain weights revealed significant differences F(3,36) = 3.837, P = 0.0176 among groups, and the post hoc PLSD test showed significantly lower brain weights in the MPb vs. C (P < 0.0076) and in the MPb vs. M (P < 0.0044) comparisons (Fig. 2).
Body weights of newborn rats
60.3 ± 20.5
(P < 0.05,
Brain cortex Lead concentration in the cerebral cortex was significantly different among groups (Kruskal–Wallis test, P = 0.0001). Lead concentration in the cerebral cortex was increased in both of the two groups of rats exposed to lead (37.7 ± 9.2 and 35.4 ± 5.7, respectively). These values were significantly different (P < 0.05) from those of the malnourished animals (M) 12.9 ± 2.6. Finally, the C group had lead levels of 23.6 ± 3.6 (Table 1).
Striatum The average values observed in the striatum of newborn animals (P0) is shown in Table 1. As we can see, there is a statistically significant increase (Kruskal–Wallis test P = 0.006 followed Mann–Whitney U test, P < 0.05) in lead concentrations in the CPb (43.3 ± 4.9) and in the MPb (38.5 ± 14.9) group when compared with the groups well-nourished and lead-free (C) and lead intoxicated (Pb) groups (18.4 ± 4.0 and 13.5 ± 3.5, respectively).
Cerebellum Lead intoxication in the hippocampus of newborn rats is shown in Table 1, and was significantly different among groups after Kruskal–Wallis tests, (P = 0.007); as shown, there are average lead levels of 25.0 ± 3.5 in C animals (without malnutrition and lead intoxication), whereas results obtained in M (malnourished and free-of-lead) animals were of 17.5 ± 5.0. In animals well-nourished and intoxicated with lead, the value was 53.6 ± 8.5 that is statistically significant compared to C and M groups (P < 0.05). Finally, the lead average value of the malnourished
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The two-way ANOVA revealed significant differences F(3,20) = 49.10, P < 0.0001 in the body weights among groups, and the post hoc PLSD test showed significantly lower (P < 0.001) body weights for the M and MPb groups of newborn rats when compared with the C and Pb groups. Moreover, the MPb group showed a significantly lower (P < 0.001) body weight than the M group (Fig. 3).
average value was Mann–Whitney U test).
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Lead concentrations in newborn-rat brains
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Hippocampus The lead concentration in the hippocampus of newborn rats were significantly different among groups, after the Kruskal–Wallis test, (P = 0.029) (Table 1). Likewise, the observed mean lead levels in C animals were 28.0 ± 6.3, while Pb animals averaged 62.5 ± 9.5 (P < 0.05). M animals showed an average value of 32.1 ± 9.9, while in MPb animals the
Figure 2 Dam’s brain weights measured immediately after delivery. C: animals under the normal diet without lead intoxication; Pb: rats under the normal diet and intoxicated with lead; M: malnourished rats without lead intoxication; and MPb: malnourished and lead-intoxicated rats. The results are expressed as means ± SEM of 10 animals per group. Oneway ANOVA followed by Fisher’s post hoc test. *P < 0.05 vs. C and M groups
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Figure 3 Newborn body weights measured immediately after birth. C: animals under normal diet without lead intoxication; Pb: rats under normal diet and intoxicated with lead; M: malnourished rats without lead intoxication; and MPb: malnourished and lead-intoxicated rats. The results are expressed as means ± SEM of six randomly selected animals per group. Two-way ANOVA followed by Fisher’s post hoc test. *P < 0.05 CPb vs. C, **P < 0.05 M different of CPb and C and †P < 0.05 MPb different from all groups.
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25.0 45.1 43.3** 20.4 16.1 18.4 Striatum
and lead intoxicated group was 51.5 ± 12.3, with significant differences when compared with C and M groups.
Hippocampus LP levels in the hippocampus of newborn rats are shown in Table 2. The results are expressed as fluorescence units per milligram of protein, the basal LP was of 23.5 ± 2.5 (control, group), likewise for the malnutrition (M) group the results are 49.3 ± 14.5. Analysis of hippocampal tissue revealed a significant increase of LP (P < 0.05) in the M and MPb groups when compared with the C and CPb groups. Lead intoxication effects were not found to differ significantly between the C and CPb or between the M and MPb groups.
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Brain cortex Cerebral cortex tissue showed an increase (P < 0.05) of LP in both M and MPb when compared with the C group (35.9 ± 3.0). There was neither an additive nor a synergistic effect of M and Pb, because there was no difference between malnourished groups (M vs. MPb). A similar difference was observed between the C and CPb groups in animals only exposed to Pb with normal diet, CPb (Table 2).
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22.4
21.1
13.0
26.3 32.1
23.1 108.5 13.8 119.7 13.4 61.0 23.7 114.3 52.3 60.5 62.5**
4.7 47.7 12.5 42.0 6.3 37.8 9.3 43.0 33.9 28.0 Hippocampus
28.6
Results are expressed in ng/g of wet tissue. The Kruskal–Wallis test followed by the Mann–Whitney U test. IQR, interquartile range; C, animals under normal diet and without lead; Pb, rats fed normal diet and exposed to lead; M, malnourished rats without lead exposure; and MPb, malnourished rats exposed to lead. Significant differences **P < 0.05 compared to C and M groups and *P < 0.05 compared to the M group.
70.4 55.5 51.5*
41.3 12.7 38.5**
35.4*
30.7
22.3
4.4 286.7 4.4 88.0 2.1 185.4 12.8 100.0 42.2 38.6 60.3**
1.8 148.3 3.6 46.2 0.5 45.8 0.2 53.6 27.7
Median IQR Median IQR Median Mean
C
Table 1 Lead concentrations in newborn-rat brains
Min Max
Mean
CPb
Min Max
Mean
Median
M
IQR
Min Max
Mean
MPb
IQR
Min Max
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Striatum Similar effects to those found in the cortex were observed in the striatum tissue; both the M and the MPb groups showed a significant increase (P < 0.05) of LP when compared with the C and CPb groups (Table 2). Cerebellum LP in the cerebellum showed a statistically significant increase (P < 0.05) with M both alone and in combination with lead (MPb), when compared with the C group (60.5 ± 3.2) (Kruskal–Wallis test followed by Mann–Whitneys U test). In addition, the post hoc Mann–Whitney U test showed no differences in LP levels in C vs. CPb (Table 2). Interestingly, for this region, a significant difference was found between the group M and group M/Pb (P < 0.05), indicating a significant interaction between Pb and M.
Discussion Our findings demonstrated heterogeneous effects on the concentrations of both LP and Pb measurements in four neonatal brain areas in response to protein restriction and lead intoxication during the gestational period. Both agents produce a significant reduction of body weights and differential oxidative damage in the brain, but the significant increase of the Pb concentrations and LP is more evident in the four newborn
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30.7 43.9 45.2
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Median Mean
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brain regions analyzed. During gestation the dams water intake was similar in the four groups (C, CPb, M, and MPb); however, a significant reduction in food intake was found in the M and MPb groups; in this group we also found a reduction in the body weight. It has been reported that chow consumption is different in the group of rats under low-protein diet, as a result of endocrine signals.36 Malnutrition (M) during the pre- and postnatal periods affects a large number of children worldwide, resulting in biochemical, physiological, and anatomical changes in the central nervous system that are associated with cognitive and behavioral impairment during childhood and adolescence.36 In animals, the protein content of the diet plays an important role in antioxidant mechanisms in the brain, and malnutrition produces an overproduction of reactive oxygen species (ROS) that lead to membrane oxidation and thus to an increase in LP due to changes in oxidant and antioxidant levels in the plasma.37 Protein malnutrition may lead to an increase in oxidative damage by diminishing antioxidant defenses of the brain.26,27 On the other hand, intrauterine exposure to a mixture of environmental pollutants has become a major health concern, because it could adversely affect the development trajectory during pregnancy.38 Lead exerts its most severe effects on the developing brain, which can be attributed to the intense cell proliferation and synaptogenesis which takes place in the various more sensitive brain areas such as the hippocampus, frontal cortex, cerebellum, and striatum.15 In fact, those areas also accumulated more lead, among the various brain regions examined (article by Villeda-Hernandez et al.). Lead differential distribution among brain regions may reflect, in turn, vascularization of the different brain regions. Furthermore, one of the important mechanisms of action of environmental chemicals may involve ROSinduced oxidative stress. 39 In the brain, chronic exposure to lead would predominantly affect two specific protein complexes: protein kinase C and the N-methyl-D-aspartate (NMDAR) subtype of the glutamate receptor. There is a hypothesis that NMDAR-receptor-dependent and protein synthesisdependent long-term potentiation are important targets for neurotoxicity induced by Pb.40 Both factors, Pb intoxication and M with only a 7% of casein, during gestation, produced increased oxidative damage to lipids and proteins.29,30 Therefore, it is necessary to understand the consequences of these adverse effects during brain development and the resulting functional impairments such as memory disturbance, learning deficits, and behavioral problems. Populations with limited economic resources may also have nutritional deficits that make them uniquely
cte
16.4
49.8 91.0 94.3**
57.6 57.7*
92.5 97.2**
20.3 102.6 16.8 87.3 27.2 64.9 17.9 173.1 67.1 33.5 49.3
20.2 34.8 28.5 47.3 34.8 67.0 50.2 72.5 11.3
Median IQR
Min Max
Results are expressed in FU/mg of protein. The Kruskal–Wallis test followed by the Mann–Whitney U test. IQR, interquartile range; C, animals under normal diet without lead intoxication; Pb, rats under normal diet and intoxicated with lead; M, malnourished rats without lead intoxication; and MPb, malnourished and lead-intoxicated rats. Significant differences, *P < 0.05 vs. C, **P < 0.05 vs. C and CPb and †P < 0.05 vs. all other groups.
126.4 171.0 161.4†
54.9 104.5 118.2**
35.7 86.4 78.6*
22.4 84.5 84.3**
71.5 139.3 34.4 76.2 63.3 145.9 65.8 111.4 18.2
IQR 6
C
Table 2 Lipid peroxidation levels in newborn-rat brains
Mean
CPb
IQR
Min Max
Mean
Median
M
46.2
IQR Median Mean
MPb
Min Max
67.2 97.4 51.3 97.2 75.3 228.8 78.9 253.1
Prenatal malnutrition and lead intake produce brain LP levels in newborn rats
Min Max
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establishment of oxidative stress, mainly in the cerebellum, indicating its higher vulnerability to lead toxicity in newborn rats. Similar effects have been described by Mousa et al.,46 who reported increase of fetal mortality and reduced body growth and brain weight due to lead intoxication; they also found fetal cerebellar damage in cortical layers, degenerative changes including formation of clumps of Nissl granules in Purkinje and granule cells, and finally, an increase neuronal apoptosis. Further experiments are needed to explain how malnutrition and lead intoxication lead to the diminished adult brain weights; we observed since another reports show diminished brain/body weights, only after 1 month of lead exposure (100 mg/kg/day).47,48 Loss of tissue, both in the adult and developing brain as a result of Pb exposure and malnutrition, may be the result of enhanced brain cells’ death, which in turn may be reflected as a reduced brain weight. This explanation remains speculative until further experiments test that hypothesis. Likewise, pre- and postnatal level of the 4-hyrdoxynanoneal (a byproduct of LP), and the effect of oxidative stress due to combined exposure to malnutrition and Pb remains a central subject to be examined in future experiments. Studies should include the response of antioxidant enzymes, such as catalase and/or superoxide dismutase, which have been reported to be increased during the postnatal period,15 in order to determine whether the effects of oxidative stress correspond to the time of neuronal differentiation in each brain region, and if they correlate with age-related changes in the capacity of the antioxidant system.
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susceptible to Pb intoxication.18,41 Pb is naturally present in nature; however, most of the high levels found in the environment come from human activities. Lead-induced enhancement of LP and redox imbalance in the brain are possible mechanisms responsible for brain damage. 27 The central nervous system is most sensitive to Pb exposure during early development due to rapid cell proliferation and migration, axonal growth, synaptogenesis, and neurotransmitter systems such as the GABAergic interneurons.42 In addition, exposure of animals to Pb has disrupted hippocampal development, with deficits in learning and memory capacities.43 We used a low dose of Pb during the gestational period when the immature rat brain is highly vulnerable. In a similar study, Baranowska-Bosiacka et al. 24 found that rats were highly vulnerable during the perinatal period to Pb exposure at a 10 μg/dl concentration, which resulted in damage to the adult hippocampus. They concluded that oxidative stress is one of the possible mechanisms disturbing cellular metabolism in this structure. During intrauterine malnutrition, female offspring present an increased superoxide production, maybe catalyzed by nitric oxide synthase, whose activity in the hippocampus, the cerebral cortex, and the cerebellum is inhibited by lead exposure to an extent that depends on its concentration and the duration of exposure.44 The lead concentration (ng/g of wet tissue) in C and MPb groups increased significantly in the four areas analyzed (Table 1), suggesting brain hypomyelination due to Pb intoxication and M during pregnancy. These concomitant effects were observed in the brain and the cerebellum in rats of 30, 60, and 120 days of age when Pb was administered on postnatal days 1–15 (P1–P15) by daily intraperitoneal injections of 10 mg lead nitrate/kg body weight.45 Malnutrition resulted in a three-fold higher LP rate in the hippocampus in the M vs. the C group. Nevertheless, the combination of both lead intoxication and M (Table 2) showed only a two-fold increase in LP compared with C, indicating an interaction between the two treatments. Previous malnutrition reports from Feoli et al.29,30 employing a 7% casein diet, demonstrated the damaging consequences within the cerebellum and the cerebral cortex of oxidative stress, especially damage of macromolecules and the increased levels of LP. These authors also reported a significant decrease in total antioxidant levels in the cerebral cortex of protein-malnourished rats. Our data indicated that prenatal protein malnutrition with a 6% casein diet during pregnancy increased the oxidative damage to brain lipids. On the other hand, when this treatment (water containing 160 ppm lead) was administered to animals exposed to the malnutrition model, the results showed the
Prenatal malnutrition and lead intake produce brain LP levels in newborn rats
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Acknowledgements This study was supported by DGAPA-UNAM and CONACYT. The authors wish to thank A. Aguilar and M. García for technical assistance. We are grateful to M. Salas and S. Castro-Chavira for their invaluable suggestions on the manuscript and to D. Pless for proofreading.
Disclaimer statements Contributors None. Funding None. Conflicts of interest None. Ethics approval None.
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