Acute neurotoxic effects of high dose methylmercury (MeHg) in humans have been well documented in the scientific literature. However, low dose effects are less well described. This study was designed to evaluate the effects of low dose MeHg (<100 nM) on human brain cells in a tissue culture model. Neuroblastoma cells (SH-Sy5y) were used in the cell culture model to study low dose effects of MeHg on cell growth, cell survival, oxidative stress and the phosphorylation of tau protein, as a measure of potential markers of cellular events associated with tauopathies. When cells were incubated in culture with MeHg (50 nM and 100 nM), there were significant decreases in cell viability as well as a significant increase in reactive oxygen species. In addition, the level of phosphorylated tau was significantly increased following treatment at both 50 nM and 100 nM MeHg, as compared to controls. Pretreatment of neuroblastoma cells with the antioxidant, N-acetylcysteine, as well as a calpain inhibitor, MDL- 28170, significantly attenuated the effects of MeHg (50 nM and 100 nM) on cell viability as well as on tau phosphorylation. Furthermore, MeHg has also been reported to alter glutamate homeostasis in the neuronal environment, resulting in excitotoxicity. Exposure of cells to a combination of MeHg (50 nM) and glutamate (1 mM) resulted in a greater decrease in cell viability as well as a greater induction in tau phosphorylation, as compared to exposures with MeHg and glutamate alone. MK-801 (4 muM), an NMDA receptor antagonist, and the intracellular calcium chelator, BAPTA-AM, both significantly inhibited tau hyperphosphorylation and protected cells from the effects of combination exposures to glutamate and MeHg. This study suggests that MeHg may increase the cellular response to glutamate thereby enhancing its excitoxicity. The overall conclusion of this work is that, low dose MeHg toxicity in neuroblastoma cells may be related to its induction of tau phosphorylation through an induction of oxidative stress, alteration in intracellular calcium levels as well as activation of mu-calpain and that blockade of this pathway may produce protective effects against MeHg induced neurological effects / acase@tulane.edu

Investigation of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD or dioxin) response in HuE-1 and SCC-12F keratinocyte cell lines revealed differential responses. Exposure of SCC-12F keratinocytes to dioxin produced a cytochrome P450 1A1 (CYP1A1) response, assayed by ethoxycoumarin-o-deethylase activity (15-35 pmol/mg protein/minute at 1-100 nM TCDD). Neither HuE-1 keratinocytes or HEK 293 cells (human embryonic kidney) produced this response (5 pmol or 1 pmol/mg protein/min for HuE-1 and HEK, respectively at 100 nM TCDD). HuE-1 cells possess truncated ARNT protein, which could cause lack of dioxin signaling in these cells. Transfection of HEK 293 cells with a CYP1A1 promoter construct, followed by dioxin exposure, induced reporter gene activity 2-3 fold, indicating these cells responded to dioxin but the endogenous CYP1A1 gene was defective Transforming growth factor beta (TGF-$\beta$), upregulated in keratinocyte culture medium by trans-RA exposure (683.2 pmol vs. 157 pmol in control), was suppressed by TCDD (41 pmol). The dominant TCDD effect was due, at least in part, to TCDD-mediated loss of trans-RA binding to its receptor (RARs) (Kd = 0.13 nM, 1.8 fmol binding sites vs. Kd = 0.13 nM, 4.3 fmol binding sites in control). Loss of binding affected trans-RA-mediated transcription, determined by transfection with a retinoic acid responsive reporter plasmid (trans-RA reporter activity = 48.1% of control, TCDD activity = 164% of control, TCDD + RA activity = 171.3% of control) Investigation of epidermal growth factor receptor (EGFR) production in HuE-1 keratinocytes revealed RAR-$\alpha$ suppressed EGFR transcription, determined by a luciferase reporter assay in absence of added ligand in every case (5.4% control). When cells were confluent and differentiating, a second RAR isoform, RAR-$\gamma ,$ was less effective at suppressing EGFR production (93% of control). When cells were actively proliferating, both RAR-$\alpha$ and RAR-$\gamma$ appeared equally effective in this action (23% vs 28% of control, for RAR-$\alpha$ or RAR-$\gamma ).$ Examination of RAR protein in HuE-1 cells revealed RAR-$\gamma$ protein was always present. RAR-$\alpha$ was only detected when added exogenously via an expression plasmid. This revealed the required up-regulation of RAR-$\alpha$ to aid cells through differentiation, while RAR-$\gamma ,$ present in high abundance, appears ineffective in mediating differentiation / acase@tulane.edu

LT is an AB5-type enterotoxin (consisting of one A-subunit and five B-subunits) produced by some strains of Escherichia coli and is responsible for the characteristic secretory diarrhea associated with this organism. LT and related toxins are also known to have powerful mucosal adjuvant activity for co-administrated protein antigens. The 28 kDa A-subunit has an ADP-ribosyltransferase that is responsible for ADP-ribosylating the GTP-binding protein Gsalpha found on the cell membrane of eukaryotic cells, leading to the subsequent activation of adenylate cyclase and increasing intracellular levels of cAMP. The 55 kDa B-subunit facilitates the entry of the LT through its binding to the host cell membrane receptor, GM1. In addition to ADP-ribosylation of Gsalpha, LT also undergoes auto-ADP-ribosylation, transferring ADP-ribose from NAD to one or more of its own arginine residues. The role of auto-ADP-ribosylation of LT in the toxicity and adjuvanticity of this molecule is currently unknown. In this study we used Quadrupole tandem mass spectrometry to identify the arginine residues in the A-subunit that had been auto-ADP-ribosylated. Using site directed mutagenesis, LT mutants were constructed that corresponded to the nine arginine residues where the modification occurred (R18G, R25G, R33G, R129G, R138K, R141K, R148K, R151G & R192G). Each mutant was assessed for its structural integrity, sensitivity to proteolytic digestion, GM1 binding, enzymatic activity and auto-ADP ribosylation ability when compared with native toxin. LT mutants show different level of modification, activity, toxicity and adjuvanticity as compared with the native toxin / acase@tulane.edu

Lead poisoning of young children has been deemed the number one environmental threat to America's young children. It is estimated that 890,000 children have levels high enough to effect their ability to learn$\sp{62}.$ While levels have decreased dramatically over the past 15 years, African-American children living in poverty in inner cities continue to be at particular risk for lead poisoning This study was undertaken to determine the prevalence of lead poisoning in high risk children residing in the City of New Orleans from 1993-1995 and to examine the relationship between elevated blood lead levels and sociodemographic and environmental characteristics. A model, at the census tract level, identifying the demographic and environmental factors associated with elevated blood lead levels was developed and used to evaluate whether the variables recommended for use by CDC effectively predict elevated blood lead levels The study confirmed that lead toxicity of poor, minority children in New Orleans is a severe problem. Overall, 38% of children had elevated levels ($\ge$10 $\mu$g/dL), 20% had levels $\ge$15 $\mu$g/dL. Eleven percent had levels $\ge$20 $\mu$g/dL, twice what is considered safe by the CDC. Furthermore, the study showed that not all poor, minority children are affected equally. Analysis by geographical unit, both clinic and census tract, show areas of greatest risk; in some parts of the city more than half of the children screened had elevated levels The geometric mean blood lead level was 7.85 $\mu$g/dL and levels remained constant over the three year study period. This is substantially higher than levels reported from the National Health and Nutritional Examination Survey, a cross-sectional survey of the U.S. population Distinct patterns by age were noted. Children three to five years had significantly (p $\le$.01) higher geometric mean blood lead levels than their younger counterparts and levels continued to stay elevated with increasing age. Surprisingly, children five to six years had the highest geometric mean levels Percent of houses vacant, median soil lead level and median income were consistent predictors of elevated lead levels. Percent of houses built prior to 1950 was not a significant predictor is any of the models run. This has important policy implications. CDC guidelines place heavy emphasis on the use of this variable to estimate lead-exposure risk in a geographic area$\sp{62}.$ However, in cities like New Orleans with a large stock of old houses and a cross-section of people living in them, age of housing alone is not a good surrogate for condition of housing. The variable percent of houses vacant may more accurately reflect the condition of the housing thereby being better able to estimate lead-exposure risk The results of this study may be helpful to other communities responding to CDC recommendations to tailor screening guidelines to local concerns / acase@tulane.edu

Two human cell lines cultured in vitro, a pre-B cell FLEB14 and a B-cell AHH-1 were treated with different concentrations of 5-azacytidine (5-azaC), N-methyl-N$\sp\prime$-nitro-N-nitrosoguanidine (MNNG), 1-$\beta$-D-arabinofuranosylcytosine (araC) and potassium dichromate $\rm(K\sb2Cr\sb2,O\sb7),$ to induce chromosomal aberrations. Cytotoxicity induced by these genotoxins was evaluated. Chromosomal abnormalities detected by G banding at cytogenetic level included: chromosome gaps, chromosome breaks, translocations, rings, additions, marker chromosomes, figures (multibranched chromosomes) and isochromosomes FLEB14 cells treated with the hypomethylating agent 5-azaC gave unusual chromosome abnormalities involving preferentially the pericentromeric region of chromosome 1. These abnormalities are similar to the cytogenetic diagnostic features reported in patients with immunodeficiency, centromeric heterochromatin instability, and facial anomalies (ICF) syndrome. The evidence indicates that the chromosome heterochromatic regions involved in these aberrations are normally hypomethylated in cultured ICF lymphoblasts. Aberrations seen in this study have also been frequently reported in breast cancer. Preferential formation of chromosome 1 anomalies involving the vicinity of the centromere was also seen in AHH-l cells treated with 5-azaC, but not so frequently as in FLEB 14 cells. The preferential rearrangement of the pericentromeric heterochromatin of chromosome 1 in FLEB 14 cells was induced with 5-azaC and not detected with MNNG, araC, or $\rm K\sb2Cr\sb2O\sb7,$ which alter DNA in various ways but do not cause hypomethylation. Thus DNA hypomethylation may be the common factor that links the induction of a distinct set of chromosome aberrations in FLEB14 cells in this study with similar chromosomal abnormalities in ICF syndrome cells and breast cancer / acase@tulane.edu

No description available.

Cardiac hypertrophy is a common consequence of many cardiovascular diseases. It is often a transition to heart failure. Although oxidants have been indicated to mediate heart failure, whether they induce hypertrophy and how they are able to induce hypertrophy are still unknown. The objective of this dissertation is to investigate the underlying molecular and cellular mechanisms of oxidant-induced heart hypertrophy. My hypothesis is that H₂O₂ induces cardiomyocyte hypertrophy through activating specific signaling pathways. First, signaling pathways that contribute to H₂O₂-induced cell size enlargement were investigated. In vitro kinase assay and Western Blot analysis were conducted to examine activation of PI3K and p70S6K1. We found that H₂O₂ is able to activate PI3K and p70S6K1 in a time- and dose-dependent manner. Inhibitor studies indicate that the activation of PI3K is upstream of p70S6K1. Both PI3K and p70S6K1 inhibitors are able to abolish H₂O₂ induced cell size enlargement and protein content increase, suggesting that this pathway is necessary for H₂O₂ induced hypertrophy. We also tested the activation of three branches of MAPKs: ERK, p38 and JNK. Experiments examining the possible relationship between MAPKs and p70S6K1 revealed that p38 MAPK is able to regulate p70S6K1 in part. Second, signaling pathways related to gene expression alteration associated with hypertrophy induced by H₂O₂ were investigated. This study focused on two important hypertrophy transcription factors, AP-1 and NFAT3. Using both gel shift assay and promoter reporter activation analysis, we found that H₂O₂ is a potent inducer of AP-1. This activation is downstream of ERK. NFAT3 was recently implicated to play a role in cardiac hypertrophy. Whether and how it is activated by H2O 2 is unknown. When cardiomyocytes transfected with pNFAT-luc, a NFAT luciferase plasmid with a luciferase gene under control of repetitive NFAT/AP-1 composite sites derived from the IL-2 promoter, were treated by H₂O₂, luciferase activity increased in a time and dose dependent manner. Experiments using pharmacological inhibitors demonstrated that this NFAT3 activation detected by luciferase assay is ERK and AP-1 dependent but calcineurin independent. This discovery reveals a novel mechanism of NFAT3 regulation in cardiomyocytes that can play an important role in hypertrophic gene regulation.

Tyrosine hydroxylase (TH) is regulated by the reversible phosphorylation of serines 8, 19, 31 and 40. Upon initiation of this study, serine 19 was unique due to its requirement of 14-3-3 binding after phosphorylation for optimal enzyme activity, although it has been more recently demonstrated that phosphorylated serine 40 also binds 14-3-3. To identify proteins that interact with TH following phosphorylation of serine 19, this amino acid was mutated to alanine and THS 19A was used as bait in a yeast two-hybrid system. From this, mouse derived cyclin-dependent kinase 11 (CDK11)ᵖ¹¹⁰ was identified as an interacting partner with THS19A. CDK11ᵖ¹¹⁰, formerly known as PITSLRE, is a serine/threonine kinase whose catalytic activity has been associated with transcription and RNA processing. The interaction between TH and CDK11ᵖ¹¹⁰ was confirmed using human CDK11ᵖ¹¹⁰ cDNA in a mammalian system. To further evaluate the regulation of CDK11ᵖ¹¹⁰ catalytic activity, interacting proteins were identified by liquid chromatography and tandem mass spectrometry (LC-MS/MS). Following the immunoprecipitation of CDK11ᵖ¹¹⁰ from COS-7 cells, the serine/threonine kinase CK2 was identified by LC-MS/MS. Similar to the mouse homolog, CDK11 p110 was found to serve as a substrate for CK2. To obtain CDK11ᵖ¹¹⁰ devoid of CK2, CDK11ᵖ¹¹⁰ was expressed in High Five insect cells and secreted into the media due to the presence of a honeybee melittin signal sequence encoded at the N-terminus of CDK11ᵖ¹¹⁰. After demonstrating retention of CDK11ᵖ¹¹⁰ kinase activity, it was evaluated for activity on the C-terminal domain (CTD) of the largest subunit of RNA polymerase II (pol II) since previous research suggested that CDK11ᵖ¹¹⁰ phosphorylates the CTD. However, only CK2 was found to phosphorylate the CTD in vitro. In contrast, both CK2 and CDK11ᵖ¹¹⁰ phosphorylated TH in vitro. In addition, CDK11ᵖ¹¹⁰ over-expression was observed to inhibit the interaction between TH and 14-3-3. A mechanism contributing to disruption of the interaction between TH and 14-3-3 may be due to CK2 phosphorylation of specific 14-3-3 isoforms, i.e. 14-3-3 τ. Collectively, these results imply that CDK11ᵖ¹¹⁰ and CK2 negatively regulate TH catecholamine biosynthetic activity since phosphoserine 19 of TH requires 14-3-3 binding for optimal enzyme activity and a decreased rate of dephosphorylation.

This dissertation focuses on the central hypothesis that in breast cancer cells containing the estrogen receptor-α (ER-α+) and wild-type p53, the BRCA-1 tumor suppressor gene is positively regulated by the steroid hormone estrogen and negatively regulated by Aromatic Hydrocarbon Receptor (AhR) ligands which damage DNA. In this dissertation, we demonstrate that BRCA-1 promoter activity is reduced by the DNA damaging agent Benzo[a]pyrene in breast cancer cells containing both a functional estrogen receptor and p53 pathway. In addition, our data suggests that exposure of MCF-7 breast cancer cells to estrogen stimulates transcription from the BRCA-1 5 ' flanking region, and this increase in transcription is paralleled by an increase in estrogen receptor-alpha interaction at the BRCA-1 promoter between -46 → -14 upstream of exon 1b. We report that in both untreated and estrogen-treated M CF-7 cells, a transcriptional complex, which we have termed an "Estrogen Responsive Unit" (ERU), containing AP-1, Sp1, and CREB family members, forms at the same -46 → -14 region which binds ER-α. Moreover, we show that wild-type p53 is required for estrogen induction of BRCA-1 and overexpression of a dominant-negative mutant variant of p53 can prevent this induction. Finally, we show that overexpression of wild-type p53 is able to disrupt the estrogen receptor interaction with the BRCA-1 ERU under both basal and estrogen-induced conditions while mutant p53 is only able to disrupt this interaction when estrogen is present. Taken together, these data suggest that loss of function of either the estrogen receptor-α or p53 signaling pathways may result in an inability for BRCA-1 regulation to occur and may in turn be a risk factor in the etiology of sporadic breast cancer.

A novel test system using isolated erythrocytes (red blood cells, RBCs) and arsine (AsH₃ gas dissolved in aqueous solution was characterized which allows for the quantitation of RBC exposure to AsH₃ in vitro and for in vitro study of the toxicity of AsH₃. AsH₃ was found to be rapidly and strongly associated with RBCs. Toxicity, measured as hemolysis, was time- and dose-dependent and exhibited a lag phase of about 30 minutes in both dog and rat RBCs. Hemolysis of dog RBCs was completely blocked by carbon monoxide preincubation and was reduced by pure oxygen. Sodium nitrite induction of methemoglobin (metHb) in intact rat RBCs decreased hemolysis, confirming the importance of hemoglobin in the mechanism of AsH₃-induced hemolysis. Spectrophotometric studies of the reaction of AsH₃ with purified dog hemoglobin revealed that AsH₃ reacted with reduced and oxygenated hemoglobin (HbO₂) to produce metHb and degraded Hb, characterized by gross precipitation of the protein. AsH₃ did not alter the spectrum of deoxyHb and did not cause degradation of metHb in oxygen, but bound to and reduced metHb in the absence of oxygen. Oxidative reactions as the putative cause of hemolysis were investigated. AsH₃ caused a slight decrease in cellular glutathione levels in dog RBCs, but only after hemolysis had reached maximum levels. Hydrogen peroxide (H₂O₂) was detected in aqueous solutions containing AsH₃ and HbO₂ or AsH₃ alone but not in intact red blood cells or lysates suggesting that cellular defenses were adequate to detoxify the amount formed. Additionally, high activity catalase or glutathione peroxidase added to solutions of HbO₂ and AsH₃ had little effect against AsH₃-induced damage. The differences between the visible spectra of HbO₂ treated with either AsH₃ or H₂O₂ suggest that two different degradative processes occur. Superoxide anion was not detected in AsH₃ mixtures and superoxide dismutase did not affect AsH₃-induced HbO₂ damage. Other antioxidants, mannitol, DMSO, ascorbate, and glutathione, had no effect against HbO₂ damage. These results indicate that the superoxide anion, the hydroxyl radical, and H₂O₂ are not responsible for AsH₃-induced HbO₂ damage and the subsequent hemolysis. A mechanism by which an arsenic species is the hemolytic agent is proposed.