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 affects migratory properties of neurons and their differentiation, axon genesis, and neuronal survival (Erlander and Tobin, 1991). In the mature nervous system, Glu is pivotal in neuroplasticity, in which there are use-dependent changes in synaptic efficacy as well as alterations in synaptic structure (Tsai et al, 1995; Kristensen et al, 1993, Scimemi et al, 2005). Memory generation and cognitive functions depend on these activities. Persistent or overwhelming activation of Glu-gated ion channels can cause neuronal degeneration via necrosis or apoptosis (Loup et al., 2000). Neuronal ''excitotoxicity," is a described phenomenon linked to the final common pathway of death of neurons in described disorders including Huntington's and Alzheimer's diseases, amyotrophic lateral sclerosis (ALS), fragile X syndrome, the most common form of inherited mental retardation, and some autistic attributes that also result from synaptic inhibitions associated with the [GABA.sub.A] receptors/ligand interactions culminating in behavioral dysfunctions, strokes (Loup et al., 2000, Peng et al., 2004, Houser and Esclapez, 2003. Narahashi et al., 1994) and may play an important role in the etiology of schizophrenia (Tsai et al., 1995, Kristensen et al., 1993).

The subunit composition and stoichiometry of native [GABA.sub.A]-receptor subtypes however remain unknown. Immunoperoxidase staining techniques reveal regional and cellular distribution of seven major subunits ([alpha]1, [alpha]3, [alpha]5, [beta]2, 3, [gamma]2, [delta] ) expressed in adult rat brain and have been allocated to identified neurons (Collins et al., 2006). A cloned a-subunit isoform ([alpha]6), which also confers unique pharmacology to recombinantly expressed [GABA.sub.A] receptors, is only expressed in a single neuron subtype- the cerebellar granule neuron (Scimemi et al., 2005). A combination of [alpha]-, [beta]-, and [gamma]- subunit variants are required in functional heterologous expression systems; for example the [gamma]2- subunit is essential for the receptor to express a classical benzodiazepine site. Thus functional and morphologically diverse neurons have been characterized by a distinct [GABA.sub.A]-receptor subunit repertoire. These data provide the basis for a functional and/or brain dysfunctional analysis of [GABA.sub.A]-receptor subtypes of known subunit composition that may reveal the path for yet to be substantiated therapeutic approaches relying on the development of subtype-selective drugs (Erlander and Tobin 1991; Olsen and Tobin 1990, Nakanishi, 1992, Vandenberg et al., 1992, Luddens and Wisden 1991).

Albeit, mercury is recognized as an environmental teratogen that selectively affects the nervous and other systems of the body. Various investigations have attempted to establish a correlation between mercury level in humans and toxic reactions in the nervous system (Olsen and Tobin 1990, Nakanishi, 1992, Vandenberg et al., 1992, Luddens and Wisden 1991, Philbert et al., 2000, Nierenberg et al., 1998). Contact with mercury at the time of neuronal networking has a profound neurotoxic effect on growing embryos particularly during organogenesis (Urbach et al., 1992). Mercury is a metal universally found in nature in the air, water, diet and other environmental pollutants that are public health hazards to which expecting mothers are constantly exposed. It has the potential at high doses to cause DNA damage to the growing fetus mainly by interacting with functional sulphydryl groups and enzymes in cells and thus influencing several metabolic pathways including cell cycle progression and/or apoptosis. Mercury grossly affects most genes involved in immune responses and induces various physical deformities: cleft lip and palate, rib defects, syndactylies, and abnormal skeletal calcification. Micrognathia and clubfeet are common during mercury intoxication in the fetal period. It inhibits in vitro microtubule formation and protein synthesis in neurons, alters membrane activity, and disrupts DNA synthesis (Fig 2). Mercury impairs mitosis and interferes with neuronal migration within the cell. Low levels of Hg[Cl.sub.2] or phenyl mercuric acetate induce abortion, growth retardation, and generate subcutaneous edema, excencephaly and anophthalmia (Chan 1998; Urbach et al., 1992, Goyer, 1996). Severe neurogenic pain syndrome develops in mercury neuropathy into a severe motor pain portraying signs and symptoms of both axonal degeneration and Guillain-Barre-like illnesses in humans (Adams et al. 1983, Urbach et al., 1992).

Intoxications simulating real ALS conditions are associated with mercury exposure; these individuals present a range of neurological symptoms from tremors, insomnia, polyneuropathy, paresthesias, emotional lability, irritability, personality changes, headaches, weakness, blurred vision, dysarthria or speech impairment, slowed mental responses ranging from insomnia, forgetfulness, and loss of appetite, as well as mild tremor that may be misdiagnosed as psychiatric illness to unsteady gait in movement (Urbacb et al., 1992, Goyer, 1996). Despite such presentations, it has proved difficult to measure the threshold for reference dose (RFD) for mercury: the lowest dose tolerated by humans without any side effects with highest consideration to natal periods (Stem 1993). This is primarily due to the poorly understood individual susceptibility to mercury.

Humans with unique major histocompatibility complex (MHC) antigens are prone to develop characteristic autoallergies/immunity on exposure to mercury; but the general effect seen phenotypically as well as psychological abnormal behavioral deficiencies associated with neural attacks are not well explained and investigated. Here we attempt to show relative expressions of genes involved in brain homeostasis by measuring in vitro receptors expression levels in human liver Hep[G.sub.2] cell-lines that are exposed to low levels of mercury [l-3[micro]g/mL]; we explore possible mechanisms leading to disturbances in brain homeostasis in humans during exposure to this metal at embryogenesis. It is our hypothesis that mercury exerts dose-related disruptions through its selective effects on genes that have influence on time-dependent neuronal network formation and thus induce variations in the severity of diseases in susceptible individuals. We used Affymetrix oligonucleotide microarray studies to find out the relative effects of low doses of mercury on chromosomes that express genes influencing human behavior, the Glycine Alpha-3, -6 GABA-A Chloride and Glutamate-gated Channel Receptors.

MATERIALS AND METHODS

Cell culture and Harvesting: Standard solutions of 10, 20 and 30 [micro]g/mL mercury concentrations in RNAse-free phosphate buffered solution pH 7.3 were prepared from stock solution of 10,000 [micro]g/mL [in 10% [HNO.sub.3]. Tenfold dilution in RPMI growth medium, supplemented with 10%-15% Fetal Bovine Serum [FBS] and 1% penicillin-streptomycin were then prepared for culturing [HepG.sub.2] cell-line previously kept under liquid nitrogen. Cells were incubated for a total of 48 hours at 37[degrees]C in a 5% C02-humidified environment. After the first 24 hours cells were washed in appropriate media and further incubated for 24 hours to achieve approximately 95% confluence. RNEasy kits (Qiagen) were used to isolate and purify RNA from the test and control HepG2 cells. Samples were initially lysed and homogenized in the presence of a highly denaturing guanidine isothiocyanate (GITC)-containing buffer. Addition of equal volumes of ethanol provides appropriate binding conditions, the sample was then applied to an RNeasy mini column where the total RNA binds to the membrane and contaminations are efficiently washed away. High quality RNA was then eluted in 30 [micro]l-100 [micro] of RNAse free deionized water. Concentration of extracted RNA was computed based on equivalency of 40 [micro]g/mL of RNA per mL in RNAse free deionized water taking into account the amount of RNAse free deionized water used for the final elution (between 30-100 [micro]l) dependent on amount of extract. Optical readings at [A.sub.260] and [A.sub.280] nm Absorbance (A) of RNA extracts were carried out using UV/VIS/NIR spectrophotometer Lambda 20 (Perkin Elmer) as previously described (Ayensu and Tchounwou2006).

Probe Array Scan and Catching the Microarray Image: We utilized Affymetrix Microarray Suite scanner having argon-ion laser equipped with a safety interlock system to scan and interrogate the Streptavidin-stained genes hybridized to ul 33 series of Affymetrix chips. Scanner was set at 2 X image scan, 3 [micro]m pixel values, at wavelength 570 nm for 50 [micro]m probe arrays with probe cells 24 [micro]m or less. For each gene, the relative expression in the exposed as compared to the control or baseline was determined for each cDNA. Internal controls employed during hybridization were kindly supplied by Affymetrix Inc. to normalize for differences in mRNA quality and efficiency of probe labeling. This procedure improves data quality used for downstream analysis. For each concentration, gene expression levels in control cells that have not been exposed to mercury were used to compare to test samples as described in Ayensu and Tchounwou 2006 and Affymetrix manual, 2002.

Statistical Analysis: Affymetrix Expression Batch Query was utilized employing the Wilcoxon's Signed Rank (WSR) test as a means for comparisons between mercury-treated (l-3[micro]g/mL concentrations) test and control HepG2 gene expressions. Stat common pairs, the intersection of the probe pairs from the baseline and experiment that are used by the Expression algorithm to make the change call were generated as signal log ratio (SLR) from the fluorescence signals emitted by the probes; SLR correlates with measure of the abundance of a transcript reflecting the change in the expression level of a transcript between a baseline noise (control) versus an experimental array. A [log.sub.2] signal ratio of 1 is equal to a fold change of 2. SLR, the quantitative change in transcript abundance estimates the magnitude and direction of change of a transcript of two arrays; see Ayensu and Tchounwou 2006 and/or Affymetrix manual, 2002 for complete experimental procedure including the hybridization and staining techniques.

RESULTS

Our results show that low levels of mercury (1-3 [micro]g/mL) has variable effects on stimulating haplotypes associated with human chromosomes 4, 5, 6, 15 and 17 that are associated with neurogenesis. There are enhanced expressions of genes located on 4pl2, 4q33-q34, 4q34.1, 5q31.3, 6q22-q23, I5q2l, and 17p13.1 while 5q35 genes were down regulated. Genes on 4pl2 haplotypes experienced increases of 6, 4 and 5 SLRs compared to controls. Haplotypes 4q33-34 and 5q34 carrying Glycine Alpha 3 GLRA3 HGNC, a GABA-alpha receptor ion-channel (receptor, alpha 3 subunit), glutamate-gated activity as well as Gamma Amino Butyric Acid A receptor. an alpha 6 (GABARA6 HGNC) activity, respectively experienced fold changes of 64. 1024, and 256 equivalent to SRL levels of 6, 10 and 8 respectively relative to the concentration ranges 1, 2 and 3[micro]g/mL of mercury exposure. Haplotype 4q is highly susceptible to mercury exposure resulting in folds higher than its effect on 5q. On the other hand the effect of mercury on I7pl3.l haplotypes was rather mild with only doubling from the background counts while expressions of haplotype 5q35 were rather downregulated with respect to background counts on concentrations of 2 and 3[micro]g/mL. No changes in levels were seen at 1[micro]g/mL mercury exposure.

On average linked genes on chromosomes 4 and 5 were up-regulated with greater than a 6- and 3-SLR differences, respectively (p [less than or equal to] 0.002) showing a clear separation in their gene expression profiles; Table 1; Figures 1, 2 and 3. Responses of these genes to mercury exposure in this study could be exploited to elucidate molecular mechanisms involved in receptors' role in mercury induced selective injury of the CNS that culminates in both physical and psychosocial disorders like Huntington's and Alzheimer's diseases, amyotrophic lateral sclerosis (ALS), fragile X syndrome, the most common form of inherited mental retardation, autistic behaviors and strokes. Genes located on chromosome 4 express GABA-A receptor subtype 3 while genes on chromosome 5 regulate expressions of GABA-A receptor subtype 6. Further analysis of mercury's role in influencing the alpha subunit levels in these molecules will be an added help to explain the role of mercury in CNS toxicopathogenesis. On probe set 207182_at chromosome 5q34, the expression levels were 3 SLR or 8 folds, 8 SLR or 256 folds, 3 SLR or 8 fold increases in expressing Gamma-aminobutyric acid (GABA) A receptor, alpha 6 (GABRA6 HGNC). This differential increases in the activities of subunits of glycine receptor alpha 3 and alpha 6 indicates the capability of mercury in influencing activities in the CNS as well as the PNS that may lead to several phenotypic expressions in behavior. By increasing receptor sites for alpha 3 and 6 it is possible to influence excitatory as well as inhibitory pathways. The behavioral consequences of such pharmacologically induced changes in the balance between inhibition and excitation are often profound (e.g., following administration of convulsant or anesthetic drugs which are known to alter GABAergic or glutamatergic neurotransmission).

Table 1: Human Chromosomes Responses to Low Levels of Mercury

PROUE SET    Gene Title (target                    Chromosome Location
ID           Dcscription)/symbol

207009_at    Paired-like homeobox 2b; PHOX2B               4p12
             HGNC; 6355 regulation of
             transcription, DNA dependent; 7275
             development; 7399 neurogenesis; 5634
             nucleus; 3700 transcription factor
             activity; 3712 transcription
             cofactor activity.

207928_s_at  Glycine receptor, alpha 3; GI.RA3            4q33-q34
             HGNC; GABA-A receptor activity; ion
             channel activity; extracellular
             ligand-gated ion channel activity,
             glycine binding (receptor, alpha 3
             subunit), glutamatc - gated chloride
             channel activity; neurotransmitter
             receptor activity; synaptic
             transmission

213963_s_at  Sin3-associated polypeptide, 30kDa;           4q34.1
             his tone deacetylase complex;
             transcription corepressor activity
             corepressor activity

215378_at    Ankyrin repeat and KH domain                  5q31.3
             containing 1; ANKHD1 HGNC; nucleic
             acid binding; cell cycle inhibitor
             pl6ink4A; immunoglobulin heavy chain
             variable domain, VH; transcription
             factor NusA, receptor, different EGF
             domains; Class II IV1HC alpha chain,
             C-terminal domain; Silencer of death
             domains, Sodd (Bag 4); Staph
             ylokinase.

207182 at    Gamma-aminobutyric acid (GABA) A               5q34
             receptor, alpha 6 (GABRA6 HGNC)

203812_at    Slit homolog 3 (Drosophila/SLIT3               5q35
             HGNC); neurogenesis, calcium ion
             binding, protein binding.

205029_s_at  Fatty acid binding protein 7, brain;         6q22-q23
             FABP7 HGNC; 6631FA metabolism; 6810
             transport; 7399 neurogenesis; 8285
             negative regulation of cell
             proliferation; 5215 transporter
             activity; 5478 Intracellular
             transporter activity; 8289 lipid
             binding.

219196_at    Secretogranin III SCG3 HGNC;                  15q21
             Transcript alignment(s)- NM_013243
             NCBl-Homo sapiens secretogranin III
             (SCGS), mRNA.

207704_s_at  Growth arrest-specific 7/CAS HGNC;            17pl3.l
             7050 cell cycle arrest; 7275
             development; 7399 neurogenesis;
             8151cell growth and/or maintenance;
             3700 transcription factor activity:
             Database ID: dlsrda_SCOP:b.1.8.1:|
             Cu, Zn superoxide dismutase, SOD.

PROUE SET ID        SLR            SLR              SLR
                Change/Fold     Change/Fold     Change/Fold
                Change (Affy)   Change (Affy)   Change (Affy)

              l[micro]g/mL Hg  2[micro]g/mL Hg  3[micro]g/mL Hg

207009_at            6/64             4/16            5/32

207928_s_at          6/64          10/1024           8/256

213963_s_at          4/16              3/8             1/2

215378_at             2/4              2/4             2/4

207182 at             3/8            8/256             3/8

203812_at             0/1            -1/-2           -1/-2

205029_s_at         7/128             5/32           8/256

219196_at           9/512            8/256           9/512

207704_s_at           1/2              1/2             2/4

SLR: signal log ratio; Affy: affymetrix.

Figure 1: Genes Up-regulated on Human Chromosomes in Responses to Low
Levels of Mercury (1-3 [micro]g/mL)

207928_s_at: Glycine receptor, alpha  4q33-q34  6/64   10/1024  8/256
3; GLRA3 HGNC

207182_at: Gamma-aminobutyric acid        5q34   3/8      6/64    3/6
(GABA) A receptor, alpha 6 (GANBRA6
HGNC)

219196_at Secretogranin III SCG3         15q21  9/512    8/256  9/512
HGNC; Transcript alignment(s)
-NM_013243 NCBI-Homo sapiens
secretogranin III (SCGS), mRNA.

[FIGURE 1 OMITTED]

[FIGURE 2 OMITTED]

[FIGURE 3 OMITTED]

DISCUSSION

GABA and glycine are probably the most important inhibitory neurotransmitters in the brain, specifically brainstem and spinal cord, respectively. Glycine is the major inhibitory neurotransmitter that participates in a variety of motor and sensory functions. Glycine is also found in the forebrain, where it has recently been shown to function as a coagonist at the N-methyl-D-aspartate [NMDA] subtype of glutamate receptor. Glycine promotes the actions of glutamate, the major excitatory neurotransmitter and therefore subserves both inhibitory and excitatory functions within the CNS. Current thoughts indicate that most GABAergic neurons in the brain are probably interneurons and are therefore uniquely able to alter the excitability of local circuits within a given brain region (Olsen and Tobin 1990). It has previously been shown that mercury chloride augments the GABA-induced current to 115% of control at 0.1 microM and to 270% of control at 100 microM and generated a slowly developing inward current carried by a variety of ions. In contrast, methytmercury suppressed the GABA-induced current. The potent stimulation of the GABA system by mercuric chloride is deemed important in mercury intoxication (Narahashi et al., 1994). Close to 30-40% of all CNS neurons utilize GABA as their primary neurotransmitter.

Animal models and humans with temporal lobe epilepsy (TLE) show alterations in relative ratios of [GABA.sub.A] receptor ([GABA.sub.A]R) subunits (Hevers and Luddens, 1998, Fritschy and Mohler 1995, 1999, Collins et al., 2006, Loup et al., 2000; Peng et al., 2004, Houser and Esclapez, 2003). These changes are complex and may involve both increased and decreased expressions of several [GABA.sub.A] R subunits (Figure 1). The functional consequences of these changes are likely to depend not only on the specific subunits that are altered but also on the cell types and cellular domains (e.g., soma and dendrites) in which the alterations occur at the location of the subunits in synaptic, perisynaptic, or extrasynaptic sites; and the resulting subunit composition of the modified receptors. It is for instance proposed that an altered expression of the [GABA.sub.A] receptor has neurophysiologic and functional consequences that might relate to the behavioral and neurological phenotype associated with fragile X syndrome (Collins et al., 2006). Interestingly, some neuropsychiatric disorders, such as anxiety, epilepsy and sleep disorders, are effectively treated with therapeutic agents that act on the [GABA.sub.A] receptor.

Many psychoactive drugs which enhance or decrease CNS excitability operate through GABAergic or glycinergic neurotransmission. Some of these drugs for example benzodiazepine and nonbenzodiazepine anxiolytic-hypnotics are routinely prescribed for a variety of disorders. Studies in how mercury affects behavioral alterations by its genetic upregulation of the alpha 3 and 6 subunits may assist in evaluating these receptor properties. The expression of the [gamma] subunit seems to be essential for conferring the modulatory actions of benzodiazepines on recombinant [GABA.sub.A] receptors and it appears that [alpha]-subunit heterogeneity determines the diversity of physiological and pharmacological responses characteristic of native [GABA.sub.A] receptors (Burt and Kamatchi 1991; Nakanishi 1992; Olsen and Tobin 1990; Vandenberg et al. 1992; Betz 1992). When coexpressed with [beta]1 subunits, for example, the widely distributed cerebral [alpha]l subunit yields a receptor with a relatively high affinity for GABA. By contrast, coexpression of the [alpha]2 or [alpha]3 subunits (with the [beta]i subunit) results in [GABA.sub.A] receptors with far lower affinities for GABA. Thus, the subunit composition of a given receptor may determine the local "response" to synaptically released GABA.

There are also multiple forms of the [beta] subunit expressed in brain (Vandenberg et al., 1992). Although their exact role in [GABA.sub.A] receptor function has yet to be determined, each contains a consensus sequence for phosphorylation by protein kinase A. There is some evidence that phosphorylation of the [beta] subunit may result in receptor desensitization seen with continuous exposure to GABA. The pharmacological differences seen between drugs, such as the benzodiazepines, which interact with [GABA.sub.A] receptors, also depend on subunit heterogeneity. Receptors which are composed of [alpha]3 subunits (together with [beta]1 and [alpha]2 subunits) yield much greater responses to benzodiazepines than do receptors which contain [alpha]1 or [alpha]2 subunits (Vandenberg et al., 1992, Betz 1992). It can be inferred that mercury's role in enhancing the effects of genes that synthesize these receptors is associated with increased inhibition or excitation of these receptors activities and cause disturbances in homeostatic mechanisms. This will be reflected in behavioral alterations in individuals translating into temporal lobe epilepsy, amyotropic lateral sclerosis, Giiillain-Barre-like illnesses in humans, Huntington's and Alzheimer's diseases and fragile X syndrome, the most common form of inherited mental retardation, and some autistic attributes that also result from synaptic inhibitions associated with the [GABA.sub.A]receptors/ligand interactions. These activities culminate in behavioral dysfunctions, strokes and may explain an important role in the etiology of schizophrenia as well as etiopathogenesis of infant type 1 diabetes seen in pancreatic beta cells destructions (Nicoletti et al., 1986; Baekkeskov et al. 1987).

The importance of glycine and glutamate is reflected in the fact that many of the therapeutically useful drugs work by selectively affecting these two neurotransmitter systems.

Conclusion

Mercury exposure at low levels (maximal of 2 and 3 [micro]g/mL concentrations) induces enhanced expression of genes located on chromosomes 4 and 5 with much increased expression of genes on chromosome 4 than that of chromosome 5 that express GABA-A subtypes 3, 6 and glutamate -gated chloride channel receptors respectively. Further analysis of mercury's role in influencing the alpha subunit levels in these molecules will be an added help to explain the role of mercury in CNS toxicopathogenesis. This differential increases in the activities of subunits of glycine receptor alpha 3 and alpha 6 indicates the capability of mercury in influencing activities in the CNS as well as the PNS that may lead to several phenotypic expressions in behavior. By increasing receptor sites for alpha 3 and 6 it is possible to influence excitatory as well as inhibitory pathways. The behavioral consequences of such pharmacologically induced changes in the balance between inhibition and excitation are often profound (e.g., following administration of convulsant or anesthetic drugs which are known to alter GABAergic or glutamatergic neurotransmission).

ACKNOWLEDGEMENTS

This work was financially supported by NIH Grant No. 5P20RR16470-02/USM-GR00978-04. (Biomedical Research Infrastructure Network), and partially supported byNIH-EARDA(IGHHDo46519-03) and the JSU-Center for University Scholars-Summer-Research Grant Award to Dr. Wellington Ayensu.

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Wellington K. Ayensu (1), (2)*, Ibrahim O. Farah (1), Raphael D. Isokpehi (1), Chung Lee (3) Paul B. Tchounwou (2)

(1) Dept of Biology, Jackson State University, Jackson, Mississippi, (2) Toxicogenomics Lab, Jackson State University, Jackson, MS.39217, (3) Department of Urology, Northwestern University Feinberg School of Medicine, Chicago, 1L 60611, US A

* CORRESPONDENCE TO DR. WELLINGTON K. AYENSU. EMAIL: [e-mail]