Perinatal exposure to lead induces morphological, ultrastructural and molecular alterations in the hippocampus
Highlights
► Pre- and neonatal Pb exposure decreased the number of hippocampal neurons. ► Lead caused ultrastructural alterations in CA1 region of hippocampus. ► Hippocampus is highly vulnerable to low level perinatal Pb exposure. ► Lead decreased BDNF level in the developing brain. ► Decreased Bax/Bcl2 ratio may protect hippocampus against Pb-induced apoptosis.
Introduction
Lead (Pb) toxicity is still a major health problem, associated with both environmental and occupational exposure (WHO, 2010). In developed countries, growing awareness of the effects of Pb on the environment and on human health has resulted in efforts to restrict the use of Pb in the production of fuels, paints, ceramic products, batteries, solder, and a variety of other consumer products (e.g. artificial turf playing fields made of nylon or nylon/polyethylene blend fibers, plastic toys and jewelry) (WHO, 2010). However, the major current source of early childhood lead exposure is still Pb-contaminated house dust (containing deteriorated Pb-based paint, Pb-contaminated soil) and tap water contaminated by leaching lead pipes. Current sources of Pb in ambient air include smelters, ore mining and processing, lead acid battery manufacturing, and coal combustion activities such as electricity generation (CDC, 2012).
Young children are particularly susceptible to Pb exposure from behavioral factors, such as frequent hand-to-mouth activities, greater gastrointestinal absorption and an immature blood/brain barrier (Lidsky and Schneider, 2003, Goldstein, 1984). The developing central nervous system is a primary target for lead. Acute Pb contamination in children (lead concentration in whole blood–Pb-B > 70 μg/dL), which is currently very rare, can have a dramatic effect on the central nervous system, i.e. brain edema, convulsions, coma, and lead encephalopathy (CDC, 2002). However, childhood Pb poisoning on a scale unheard of for decades has been detected in rural northwestern Nigeria. A total of 161 deaths in the two villages have been attributed to the incident from May 2009 to May 2010, with hundreds and potentially thousands more people becoming seriously ill (Moszynski, 2010, Dooyema et al., 2012).
Exposure to lower doses of Pb can lead to subtle non-specific disorders in brain functions—reduced perception; impaired cognition, impaired hearing and sight; even disorders in neurobehavioral functioning, including aggression (Schwartz and Otto, 1987, Bleecker et al., 2005). It has also been shown that Pb-B, even below 10 μg/dL, may be one of the factors that induces lower IQ in schoolchildren (Jakubowski, 2011, Canfield et al., 2003, Lanphear et al., 2005, Bellinger and Dietrich, 1994, Bellinger et al., 1992). Moreover, chelation therapy, which is recommended for children with Pb-B above 45 μg/dL, may reduce the amount of Pb in the organism but will not compensate for cognitive and behavioral problems resulting from Pb exposure earlier in childhood (Rogan et al., 2001).
Despite numerous studies, the precise mechanisms by which Pb exerts neurotoxic effects are not fully understood. It has been shown that this toxic metal may cause apoptosis in cultured rat cerebellar neurons (Oberto et al., 1996), hippocampal neurons (Niu et al., 2002), retinal rod cells (He et al., 2000), and PC12 neuronal cells (Sharifi and Mousavi, 2008). Apoptosis, “programmed” cell death, is an active process characterized by morphological features including chromatin condensation, cell and nuclear shrinkage, and oligonucleosomal DNA fragmentation (Sharifi et al., 2010, Cohen, 1997). It is regulated by a number of anti- and proapoptotic genes expressing homologous proteins and by enzymatic cascades. One of the gene families closely related to these regulatory pathways is the Bcl-2 family, which comprises several Bcl-2 related genes (Hockenbery et al., 1990) that promote (e.g. Bax) (Oltvai et al., 1993) or inhibit apoptosis (e.g. Bcl-2) (He et al., 2003). Apoptotic pathways can be regulated and abolished at several distinct points, especially caspase activation (Chetty et al., 2005). Caspases (being a family of Cys proteases) play an important role in neuronal cell death during development as well as after neuronal impairment. The basic mechanism includes the activation of caspase-3 (Casp-3) precursor protein by upstream signals such as the release of mitochondrial cytochrome C and the cleavage of specific aspartate residues in proteins with various structural and regulatory functions, thus leading to cell apoptosis (Cohen, 1997). It is possible that Pb might play a role in the activation of one of the aforementioned processes, ultimately leading to apoptosis. The consequences of apoptosis in Pb-induced brain damage may be reflected in observed behavioral, motor and cognitive impairments.
Although in most cases “programmed cell death” is achieved via a family of caspases, an important number of regulated apoptosis pathways are caspase-independent (Pradelli et al., 2010). Moreover, more recent data has revealed that there are also active caspase-independent necrotic pathways defined as necroptosis (programmed necrosis) (Delavallée et al., 2011). This Bax-mediated mitochondrial release of apoptosis-inducing factor (AIF) and its translocation to the nucleus promotes chromatinolysis and is a critical factor in programmed necrosis.
The pro-apoptotic effects of Pb in the rat brain have been reported (Han et al., 2007, Chao et al., 2007, Zhang et al., 2004, Sharifi et al., 2010, Liu et al., 2010, Kiran Kumar et al., 2009). However, Pb-induced necroptosis in the rat brain has not been studied yet. Moreover, only a few works have analyzed the neurotoxicity of Pb at blood concentrations considered safe for humans (below 10 μg/dL, CDC, 2007), particularly in the developing brain.
Brain-derived neurotrophic factor (BDNF) plays important roles in the proliferation, differentiation and survival of neurons during development, as well as in the synaptic activity and plasticity of mature neurons, and is critically involved in synaptic transmission in the hippocampus, as well as in learning and memory (Cohen-Cory et al., 2010). It has been shown that BDNF can also protect neurons from apoptosis (Zhang et al., 2011). Neal et al. (2010) have shown that embryonic hippocampal neurons cultured with Pb had decreased expression of BDNF.
Over the past decade attention has been focused on the hippocampus as a target for lead. The hippocampus is functionally related to vital behaviors and intellectual activities such as memory and learning that are affected by Pb, particularly in young children (Sharifi et al., 2010, Marchetti, 2003) through still undefined mechanism(s). Understanding of the mechanisms of Pb neurotoxicity may provide a basis for developing a new therapeutic strategy aimed at preventing vital behavior abnormalities induced by Pb poisoning.
Hence, the aim of this paper is to examine if pre- and neonatal exposure (Pb concentrations below a ‘safe level’ in rat offspring blood) may intensify or inhibit apoptosis or necroptosis in the developing rat brain. We studied Casp-3 activity and expression, AIF nuclear translocation, DNA fragmentation, as well as Bax, Bcl-2 mRNA and protein expression and BDNF concentration in selected structures of the rat brain: the forebrain cortex (FC), cerebellum (C) and hippocampus (H). Our microscopic examinations showed alterations in hippocampal neurons.
Section snippets
Animals
Procedures involving animals were carried out in strict accordance with international standards of animal care guidelines and every effort was made to minimize suffering and the number of animals used. Experiments were approved by the Local Ethical Committee on Animal Testing at the Pomeranian Medical University in Szczecin, Poland (approval No 30/2008).
Three-month old female (250 ± 20 g) Wistar rats (n = 6) were kept for a week in a cage with sexually mature males (2:1). All animals were allowed
Lead concentration in whole blood and brain
The Pb regimen used in the present experiment (0.1% PbAc in drinking water from the first day of gestation till weaning at postnatal day 21) caused significantly higher Pb concentrations with respect to control rats (drinking distilled water) measured at postnatal day 28 both in whole blood (Pb-B) and in all examined parts of the brain of intoxicated rats (Table 1). Lead concentrations in whole blood correlated strongly positively with Pb concentrations in the brain (FC: Rs = +0.72; C: Rs = +0.63;
Discussion
We investigated mechanisms of Pb neurotoxicity by determining the effect on apoptosis and expression of apoptosis-related genes Bax and Bcl2; Casp-3 activity and gene expression; AIF nuclear translocation, DNA cleavage into HMW fragments as well as BDNF concentration in selected structures of the rat's brain. The model of pre- and neonatal rats exposure to Pb used in this study resulted in whole blood Pb concentration in the offspring below 10 μg/dL (the level of concern for humans as defined by
Conclusion
Our data show that pre- and neonatal exposure of rats to Pb, leading to Pb-B below 10 μg/dL (the threshold of Pb-B value considered safe for people), can decrease the number of hippocampus neurons, occurring concomitantly with ultrastructural alterations (elongated, swollen or shrunken mitochondria; irregular nuclei with deep invagination of the nuclear membrane into the nucleoplasm and irregular clumping of chromatin; swollen synapses with thickened vesicles in the pre-synaptic part) in this
Conflict of interest
The authors declare that they have no competing interests
Acknowledgments
This study was supported by the statutory budget of the Department of Biochemistry and Medical Chemistry, Pomeranian Medical University.
References (73)
- et al.
Disrupted pro- and antioxidative balance as a mechanism of neurotoxicity induced by perinatal exposure to lead
Brain Res.
(2012) - et al.
Dexamethasone-induced apoptosis involves cleavage of DNA to large fragments prior to internucleosomal fragmentation
J. Biol. Chem.
(1993) - et al.
Systemic administration of lipopolysaccharide induces molecular and morphological alterations in the hippocampus
Brain Res.
(2010) - et al.
Molecular determinants of Pb2+ interaction with NMDA receptor channels
Neurochem. Int.
(2008) - et al.
Synaptic plasticity in the CA1 and CA3 hippocampal region of pre- and postnatally lead-exposed rats
Toxicol. Lett.
(1998) - et al.
Protective effects of ascorbic acid against lead-induced apoptotic neurodegeneration in the developing rat hippocampus in vivo
Brain Res.
(2007) - et al.
Lead and calcium produce rod photoreceptor cell apoptosis by opening the mitochondrial permeability transition pore
J. Biol. Chem.
(2000) - et al.
Effects of lead exposure on proliferation and differentiation of neural stem cells derived from different regions of embryonic rat brain
Neurotoxicology
(2004) - et al.
Lead-induced alteration of apoptotic proteins in different regions of adult rat brain
Toxicol. Lett.
(2009) - et al.
Lead exposure during synaptogenesis alters NMDA receptor targeting via NMDA receptor inhibition
Neurotoxicology
(2011)