Effects of exposure to low concentrations of nercury on Glycine Alpha-3,-6 GABA-A Chloride and glutamate-gated channel receptors in the HepG2 cell line in culture.

ABSTRACT

Neuronal networking in specific regions of the developing brain including the hippocampus is critically regulated by GABAergic signaling. Sequential progression in several stages of development during embryogenesis commence with first formation of functional GABAergic synapses and culminate in organized initial signals that play important regulatory roles in the growth of young neurons that lay the foundation for the normal establishment of central and peripheral networks necessary for brain activities. Normal development of the nervous system and certain forms of epileptogenesis, for instance, has a common pathway during growth of neurons and axons. This observation has led to the belief that there must be common molecular mechanisms for some aspects of normal development and epileptogenesis; indicating also that there must be some distinct paths between normality and abnormal neurogenesis. Developmental mechanisms therefore contribute to network changes associated with several CMS pathologies. This forms a useful strategy for identifying molecules that play a role in both of these processes. In the course of synapses formation exposure to xenobiotics, mercury in particular exerts maximal harm on growth patterns in the CNS and thus contributes to eventual dysfunctions in behavior at later years. Behavioral deficits reminiscent of low level mercury toxicity, that appear years after birth are difficult to be retrospectively associated with processes occurring in early developmental periods. Thus it is a challenge to decipher the molecular mechanisms underlying mercury-provoked neuropathies. We previously demonstrated through microarray analyses that exposure to mercury differentially influence activities of numerous genes including induction of cytotoxicity, apoptosis and activation of several genes in almost all human chromosomes via transcription. In this communication we hypothesize that developmental processes are influenced by specific regulatory molecules that play important roles; changes in their expression levels can lead to alterations in the signal transduction pathways influencing normal synapses formation or functions leading to pathology. We therefore used Affymetrix oligonucleotide microarray with minimal probe sets complementary to over 20,000 genes to demonstrate expression patterns of genes on human chromosomes that particularly regulate neuronal development and lead to behavioral deviations. We observed that GABAergic-associated signaling molecules, Glycine Alpha-3, -6 GABA-A Chloride and Glutamate-gated Channel receptors in HepG2 cells were highly overexpressed above background levels upon exposure to low doses of mercury (1-3[micro]g/mL). These molecules are found in distinct areas of the brain and exposure to mercury in the perinatal period can lead to the induction of high expression levels of these receptors sufficient to guide pathological neuronal networking through effects on genes expressed on several chromosomes including 4 and 5.

INTRODUCTION

Brain activities depend among others, on signaling via the GABAergic system of neurotransmitters. Approximately nineteen ([[alpha].sub.1-6], [[beta].sub.1-3], [[gamma].sub.1-3], [delta], [epsilon], [theta], [pi], and [rho] 1-3) known GABA receptor subunits form varieties of functional clusters throughout areas of the brain. These clusters are involved in generating neurotransmitters for specific brain activities (Hevers and Luddens, 1998). Among these receptors we find [GABA.sub.A] glycine-and glutamate-gated receptors forming major inhibitory and excitatory signal transducing molecules respectively in regions of mammalian brains (Fritschy and Mohler, 1999; Collins et al., 2006). At least 15 of these subunits ([alpha]1-6, [beta]1-3, [gamma]1-3, [theta], and [rho]1-2) form clusters associated with various forms of neuropathy (Collins et al., 2006; Loup et al. 2000; Peng et al. 2004; Houser and Esclapez 2003; Narahashi et al. 1994). The [GABA.sub.A]-receptors families are heterogeneously distinct structures expressed as heteromeric receptor complexes. Subunit composition of receptor subtypes determine physiological properties as well as their pharmacological profiles, thereby contributing to flexibility in signal transduction and allosteric modulation. The functional capabilities of individual receptor subunits influence the quality of signaling in different parts of the brain through formation of specific pentamers that display characteristic influence through release of neurotransmitters (Hevers and Luddens, 1998).

Yet a variety of chemicals influence and are capable of modifying the [GABA.sub.A] receptor-chloride channel complexes. Diverse forms of structurally unrelated chemicals do augment the GABA-induced chloride current, while others suppress the process. Mercury, like other heavy metals and a variety of polyvalent cations enhance or repress the current in a potent and efficacious manner. [GABA.sub.A]-mediated responses are implicated in several dysfunctional behaviors observable in anxiety state, depressive moods, epileptic episodes, insomnia, learning and memory impairments. The glutamate (Glu)-gated responses, among others lead to major excitatory responses in the nervous system (Peng et al, 2004; Houser and Esclapez, 2003; Narahashi et al. 1994). Nevertheless, the functions of Glu are much more diverse and complex. Glu plays a significant role in brain development; it ...