Pharmacology & Toxicology Department Theses and Dissertations
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The advanced degree programs at the Indiana University of Medicine Department of Pharmacology and Toxicology prepare scientists for careers across the spectrum of biomedical research. The Master of Science (M.S.) degree is a thesis research degree that gives a student the intellectual background to understand and participate in ongoing research projects. The Doctor of Philosophy (Ph.D.) degree is offered for the student who wants to pursue an independent career in research. Students with the Ph.D. degree are prepared for an academic career combining research with teaching or for a career in industrial pharmaceutical research. A combined M.D./Ph.D. degree is open to qualified individuals who ultimately seek to direct biomedical research with a clinical emphasis.
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Item Evaluation of Storage Conditions for Assessing DNA Damage Using the Comet Assay(2006-11-02T14:25:05Z) Villavicencio, Dante; Klaunig, James E.; Kamendulis, Lisa M.; Willis, Lynn R.The single cell gel electrophoresis assay (comet assay) is a useful tool for monitoring individuals who may be at risk of DNA damage and the ensuing process of carcinogenesis or other disease states. Leukocytes in blood samples provide a means of obtaining cells for use in the comet assay. However instances may arise when samples must be stored for later analysis. The present study investigated the effects of storage conditions on DNA damage in the form of strand breaks and oxidized bases in rat and human leukocytes using the comet assay. Whole blood and buffy coat samples were stored at room temperature or 4ºC for 1, 2, 24, and 48 hours or cryopreserved at -80ºC for 1 day and 1, 2, 3, and 4 weeks. The results show that the time of storage is limited if the whole blood or buffy coat samples are stored at room temperature or 4ºC. However, if cryopreserved using glycerol or DMSO as the cryoprotectant, the samples may be stored for at least 4 weeks without DNA strand breaks or oxidative damage deviating significantly from the fresh samples.Item NEUROFIBROMIN, NERVE GROWTH FACTOR AND RAS: THEIR ROLES IN CONTROLLING THE EXCITABILITY OF MOUSE SENSORY NEURONS(2007-01-03T18:34:09Z) Wang, Yue; Nicol, Grant D.; Vasko, Michael R.; Clapp, D. Wade; Cummins, Theodore R.ABSTRACT Yue Wang Neurofibromin, nerve growth factor and Ras: their roles in controlling the excitability of mouse sensory neurons Neurofibromin, the product of the Nf1 gene, is a guanosine triphosphatase activating protein (GAP) for p21ras (Ras) that accelerates the conversion of active Ras-GTP to inactive Ras-GDP. It is likely that sensory neurons with reduced levels of neurofibromin have augmented Ras-GTP activity. In a mouse model with a heterozygous mutation of the Nf1 gene (Nf1+/-), the patch-clamp recording technique is used to investigate the role of neurofibromin in controlling the state of neuronal excitability. Sensory neurons isolated from adult Nf1+/- mice generate more APs in response to a ramp of depolarizing current compared to Nf1+/+ mice. In order to elucidate whether the activation of Ras underlies this augmented excitability, sensory neurons are exposed to nerve growth factor (NGF) that activates Ras. In Nf1+/+ neurons, exposure to NGF increases the production of APs. To examine whether activation of Ras contributes to the NGF-induced sensitization in Nf1+/+ neurons, an antibody that neutralizes Ras activity is internally perfused into neurons. The NGF-mediated augmentation of excitability is suppressed by the Ras-blocking antibody in Nf1+/+ neurons, suggesting the NGF-induced sensitization in Nf1+/+ neurons depends on the activation of Ras. Surprisingly, the excitability of Nf1+/- neurons is not altered by the blocking antibody, suggesting that this enhanced excitability may depend on previous activation of downstream effectors of Ras. To determine the mechanism giving rise to augmented excitability of Nf1+/- neurons, isolated membrane currents are examined. Consistent with the enhanced excitability of Nf1+/- neurons, the peak current density of tetrodotoxin-resistant (TTX-R) and TTX-sensitive (TTX-S) sodium currents (INa) are significantly larger than in Nf1+/+ neurons. Although the voltage for half-maximal activation (V0.5) is not different, there is a significant depolarizing shift in the V0.5 for steady-state inactivation of INa in Nf1+/- neurons. In summary, these results demonstrate that the enhanced production of APs in Nf1+/- neurons results from a larger current amplitude and a depolarized voltage dependence of steady-state inactivation of INa that leads to more sodium channels being available for the subsequent firing of APs. My investigation supports the idea that regulation of channels by the Ras cascade is an important determinant of neuronal excitability. Grant D. Nicol, Ph.D, ChairItem Functions of the Unique N-terminus of a GCN5 Histone Acetylase in Toxoplasma gondii(2007-05-18T13:14:16Z) Bhatti, Micah M.; Sullivan, William J., Jr.; Chan, Edward M.; Queener, Sherry F.; Safa, Ahmad R.; Sinai, Anthony P.; Vasko, Michael, R.GCN5 is a histone acetyltransferase (HAT) that remodels chromatin by acetylating lysine residues of histones. The GCN5 HAT identified in Toxoplasma gondii (TgGCN5) contains a unique N-terminal “extension” that bears no similarity to known proteins and is devoid of known protein motifs. The hypothesis of this thesis is the N-terminal extension is critical to the function of TgGCN5. Three possible roles of the N-terminus were investigated: nuclear localization, protein-protein interactions, and substrate recognition. Subcellular localization was determined via immunocytochemistry using parasites expressing recombinant forms of TgGCN5 fused to a FLAG tag. Initial studies performed with parasites expressing full length FLAG-TgGCN5 were positive for nuclear localization. Without the N-terminal extension (FLAG-ΔNT-TgGCN5) the protein remains cytoplasmic. Additional studies mapped a six amino acid motif (RKRVKR) as the nuclear localization signal (NLS). When RKRVKR is fused to a cytoplasmic protein, it gains access to the nucleus. Furthermore, we have established the NLS interacts with Toxoplasma importin α, a protein involved in nuclear trafficking. Interaction with importin α provides evidence that the TgGCN5 N-terminal extension is involved in mediating protein-protein interactions. In order to identify additional interacting proteins, FLAG affinity purification was performed on parasites expressing full length FLAG-TgGCN5 and FLAG-ΔNT-TgGCN5. Upon comparing the results of the two purifications, proteins captured with only full length TgGCN5 may be interacting with the N-terminal extension. Full length TgGCN5 affinity purification indicates an interaction with histone proteins, two different homologues of Ada2 (adapter protein reported to interact with GCN5 homologues), and several heat shock proteins. With regard to substrate recognition, the N-terminal extension of TgGCN5 is dispensable for the acetylation of non-nucleosomal histones in vitro. However, the lysine acetylated by TgGCN5 is surprisingly unique. Other GCN5 homologues preferentially acetylate lysine 14 in histone H3, but TgGCN5 exclusively acetylates lysine 18 in histone H3 and has no activity on lysine 14. Taken together, these results argue that the N-terminal extension of TgGCN5 is critical for mediating protein-protein interactions, including those responsible for trafficking the HAT to the parasite nucleus but does not appear to be required for the acetylation of non-nucleosomal histones.Item Cellular Mechanisms Mediating the Actions of Nerve Growth Factor in Sensory Neurons(2007-08-08T15:24:37Z) Park, Kellie Adrienne; Vasko, Michael R.Nerve growth factor (NGF) is a neurotrophin upregulated with injury and inflammation. Peripheral administration of NGF causes hyperalgesia and allodynia in animals. Blocking NGF signaling reverses these effects. At the cellular level, chronic exposure of sensory neurons to NGF enhances expression the neurotransmitter, calcitonin gene-related peptide (CGRP). Acute exposure to NGF increases capsaicin-evoked CGRP release from sensory neurons in culture. Thus, NGF increases peptide release from neurons by: (1) increasing expression of peptides, and/or (2) altering their sensitivity. The increase in peptide outflow by either mechanism could contribute to development of hyperalgesia and allodynia. The signaling cascades mediating the actions of NGF in sensory neurons are unclear. Therefore, experiments were designed to determine which pathways regulate changes in iCGRP content and evoked release from primary sensory neurons in culture. The Ras/MEK/ERK cascade was identified as a possible regulator of iCGRP expression in response to NGF. To test this pathway, it was manipulated in neurons by (1) expression of dominant negative or constitutively active isoforms of Ras, (2) farnesyltransferase inhibition, (3) manipulation of the RasGAP, synGAP, and (4) blocking MEK activity. When the pathway was blocked, the NGF-induced increase in iCGRP expression was attenuated. When the Ras pathway was activated, iCGRP expression increased. These data indicate that Ras, and downstream signaling kinases, MEK and ERK, regulate the NGF-induced increases in CGRP in sensory neurons. To determine which pathway(s) regulate the increase in capsaicin-evoked iCGRP release upon brief exposure to NGF, the Ras/MEK/ERK pathway was manipulated as described above, and pharmacological inhibitors of the PI3 kinase, PLC, and Src kinase pathways were used. There were no differences observed in NGF-sensitization when the Ras and PI3 kinase pathways were inhibited, suggesting these two pathways were not involved. However, when the Src kinase inhibitor PP2 was used, the NGF-induced increase in release was completely blocked. Furthermore, the PKC inhibitor, BIM, also inhibited the sensitization by NGF. This data indicate Src and PKC regulate of sensitivity of sensory neurons in response to brief exposure to NGF. Thus, there is differential regulation of iCGRP content and evoked release from sensory neurons in response to NGF.Item Vincristine Metabolism and the Role of CYP3A5(2007-11-16T20:07:34Z) Dennison, Jennifer Bolin; Hall, Stephen D. (Stephen David), 1957-; Kamendulis, Lisa M.; Queener, Sherry F.; Erickson, Leonard C.; Wrighton, Steven A.Vincristine is metabolized by the cytochrome P450 3A subfamily of enzymes possibly including CYP3A5, a genetically polymorphic enzyme. The contribution of CYP3A5 to the metabolism of vincristine was quantified by various in vitro models: cDNA-expressed enzymes, human liver microsomes, and human hepatocytes. With these models, the major CYP metabolite of vincristine, M1, was identified and extensively characterized. The rates of M1 formation in the cDNA-expressed enzyme models were at least 7-fold higher with CYP3A5 than CYP3A4; approximately 90% of the hepatic metabolism was predicted to be CYP3A5-mediated. For human liver microsomes with high CYP3A5 expression, the CYP3A5 contribution was substantial, approximately 80%. Human hepatocytes with at least one CYP3A5*1 allele also metabolized vincristine, albeit at a slower rate (10-fold) than human liver microsomes. The CYP3A5 low-expressing hepatocytes did not metabolize vincristine. We conclude that for high CYP3A5 expressers, the majority of the CYP metabolism is mediated by CYP3A5. By in vitro/in vivo scaling with microsomes, the hepatic clearances of high CYP3A5 expressers are predicted to have a 5-fold higher hepatic clearance than low expressers. However, the role of metabolism in the systemic clearance of vincristine is unknown. To study the disposition of vincristine in vivo, a sensitive and selective LC/MS/MS assay was validated for the quantification of vincristine and M1 quantification in human plasma. Vincristine and M1 were identified and quantified in select pediatric plasma and urine samples. For future large-scale clinical studies, the vincristine and M1 concentrations in plasma will be quantified to understand the role of CYP3A5 genotype in vincristine pharmacokinetics. For patients that are CYP3A5 high expressers, the systemic clearance of vincristine may be higher than that of low CYP3A5 expressers. Thus, CYP3A5 genotype may be an important determinant of inter-individual variability in clinical outcomes.Item Electrophysiological and Pharmacological Properties of the Neuronal Voltage-gated Sodium Channel Subtype Nav1.7(2007-12) Sheets, Patrick L.; Cummins, Theodore R.; Nicol, Grant D.; Oxford, Gerry S.; Vasko, Michael R.; Schild, John H.Voltage-gated sodium channels (VGSCs) are transmembrane proteins responsible for the initiation of action potentials in excitable tissues by selectively allowing Na+ to flow through the cell membrane. VGSC subtype Nav1.7 is highly expressed in nociceptive (pain-sensing) neurons. It has recently been shown that individuals lacking the Nav1.7 subtype do not experience pain but otherwise function normally. In addition, dysfunction of Nav1.7 caused by point mutations in the channel is involved in two inherited pain disorders, primary erythromelalgia (PE) and paroxysmal extreme pain disorder (PEPD). This indicates Nav1.7 is a very important component in nociception. The aims of this dissertation were to 1) investigate if the antipsychotic drug, trifluoperazine (TFP), could modulate Nav1.7 current; 2) examine changes in Nav1.7 properties produced by the PE mutation N395K including sensitivity to the local anesthetic (LA), lidocaine; and 3) determine how different inactivated conformations of Nav1.7 affect lidocaine inhibition on the channel using PEPD mutations (I1461T and T1464I) that alter transitions between the different inactivated configurations of Nav1.7. Standard whole-cell electrophysiology was used to determine electrophysiological and pharmacological changes in WT and mutant sodium currents. Results from this dissertation demonstrate 1) TFP inhibits Nav1.7 channels through the LA interaction site; 2) the N395K mutation alters electrophysiological properties of Nav1.7 and decreases channel sensitivity to the local anesthetic lidocaine; and 3) lidocaine stabilizes Nav1.7 in a configuration that decreases transition to the slow inactivated state of the channel. Overall, this dissertation answers important questions regarding the pharmacology of Nav1.7 and provides insight into the changes in Nav1.7 channel properties caused by point mutations that may contribute to abnormal pain sensations. The results of this dissertation on the function and pharmacology of the Nav1.7 channel are crucial to the understanding of pain pathophysiology and will provide insight for the advancement of pain management therapies.Item Estrogenic Activity of the Polybrominated Diphenyl Ether Flame Retardant Mixture DE-71(2008-03-05T20:08:04Z) Mercado-Feliciano, Minerva; Bigsby, Robert M.; Klaunig, James E.; Jeng, Meei-Huey; Kamendulis, Lisa; Skaar, Todd; Sullivan, William J., Jr.Polybrominated diphenyl ethers (PBDEs) are widely used flame retardants suspected to act as endocrine disruptors. We tested the commercial PBDE mixture DE-71 and its in vivo metabolites for estrogenic activity. MCF-7 breast cancer cells culture, ERE-luciferase gene expression, 3H-β-estradiol displacement from recombinant ERα, and ovariectomized (OVX) mice served as bioassays. Although DE-71 did not bind ERα, it was able to increase MCF-7 cell proliferation and this was prevented by the antiestrogen fulvestrant. DE-71 co-treatment reduced the effect of estradiol in MCF-7 cells. In the OVX mouse (BALB/c) 3-day assay, DE-71 administered alone had no effect on uterine or vaginal tissues but when administered subcutaneously potentiated estradiol’s effect on uterine weight in a dose-dependent manner. DE-71 administered SQ to BALB/c mice for 34 days slightly increased uterine epithelial height (UEH), vaginal epithelial thickness (VET) and mammary ductal lumen area, and attenuated the estradiol-induced increase in UEH; these effects were not seen in C57BL/6 mice. DE-71 increased liver weight in BALB/c, C57BL/6 and estrogen receptor-alpha knockout (ERαKO) mice. Liver cytochrome P450 1A (CYP1A) and CYP2B activities increased 2.5-fold and 7-fold respectively when DE-71 was administered PO, but only CYP2B increased (5-fold) after SQ treatment. Six OH-PBDE metabolites were found in mice after 34-day DE-71 treatment and all were able to bind recombinant ERα. Para-hydroxylated metabolites displayed a 10- to 30-fold higher affinity for ERα compared to ortho-hydroxylated PBDEs. Para-OH-PBDEs induced ERE-luciferase and produced an additive effect when coadministered with β-estradiol. DE-71 was also additive with β-estradiol. At high concentrations (≥ 5x10-5 M), ortho-OH-PBDEs were antiestrogenic in the ERE-luciferase assay. In conclusion, DE-71 behaves as a weak estrogen in both MCF-7 breast cancer cells and ovariectomized adult mice. Mice strain, treatment route and duration determined if DE-71 was estrogenic. BALB/c mice are more susceptible to DE-71 effects in estrogen target tissues than C57BL/6 mice. DE-71 increased liver weight, 5%-51% depending on mouse strain and treatment regime, independently of ERα. The observations that the DE-71 mixture does not displace 3H-β-estradiol from ERα while the hydroxylated metabolites do, suggest that the cellular and tissue effects were due to a metabolic activation of individual congeners.Item Identification and functional characterization of genetic variants in the human indoleamine 2, 3-dioxygenase (INDO) gene(2008-10-13T18:43:27Z) Arefayene, Million; Flockhart, David A.; Vasko, Michael R.; Queener, Sherry F.; Safa, Ahmad R.; Wilkes, David S.Indoleamine 2,3-dioxygenase (IDO) is a rate limiting enzyme in tryptophan catabolism that has been implicated in the pathogenesis of a number of diseases. Large interindividual variability in IDO activity in the absence of stimuli and as the result of therapy induced changes has been reported. This variability has the potential to contribute to susceptibility to disease and to interindividual variability in therapeutic response. To identify genetic variations that might contribute to interindividual variability in IDO activity, we resequenced the exons, intron/exon borders and 1.3 kb of the 5’-flanking region of the INDO gene in 48 African-American (AA) and 48 Caucasian-American (CA) subjects from the Coriell DNA Repository. A total of 24 INDO variants were identified. Seventeen of these were in exons, introns, or exon/intron boundries, while seven were within 1.3 kb upstream of the translation start site. Seventeen are novel and 7 were previously identified. When transiently expressed in COS-7 or HEK293 cells the amino acid sequence change in Arg77His resulted in significant decrease in activity, and it reduced the Vmax of IDO. The Arg77His variant and the 9 bp deletion resulted in nearly complete loss of enzyme activity and a lack of detectable protein expression. The function of the Arg77His variant IDO was restored in a dose dependent manner by the heme analog hemin; but there was no associated increase in IDO protein. Cellular heme concentration was higher in cells transfected with the wild type and Ala4Thr variant constructs, but not in cells transfected with the Arg77His variant. The heme synthesis inhibitor, succinylacetone (SA), blocked IDO activity in cells transfected with Arg77His. We identified 22 putative transcription binding sites within the 1.3 kb upstream of the translation start site. Two of the SNPs were located in GATA3 and FOXC1 sites. A specific 3-SNP haplotype reduced promoter activity when transiently transfected into 2 different cell lines. We conclude that there are naturally occurring genetic variants in the INDO gene which affect both expression and activity. These results make clear that interindividual variability in IDO activity at baseline or in response to therapy may be in part due to inherited genetic variability.Item Propoxyphene, Norpropoxyphene, and Proadifen (SKF-525A) Are Mechanism Based Inhibitors of CYP3A4, CYP3A5, and CYP3A in Human Liver Microsomes(2009-03-18T18:37:57Z) Riley, Anna Ruth; Queener, Sherry F.; Jones, David R.; Flockhart, David A.; Willis, Lynn R.The purpose of this study is to determine if propoxyphene and norpropoxyphene are mechanism-based (irreversible) inhibitors of CYP3A, and to determine if propoxyphene and norpropoxyphene are reversible inhibitors of CYP3A. Mechanismbased inhibition is a type of irreversible inhibition that results from an inhibitor or its metabolite binding to an enzyme during drug metabolism, which renders the enzyme nonfunctional. Propoxyphene is an analgesic that is frequently prescribed in the United States and Europe. It is metabolized by CYP3A enzymes, and is an irreversible inhibitor of CYP3A4. The major metabolite of propoxyphene is norpropoxyphene, which has not been extensively studied for enzyme inhibition. Proadifen (SKF-525a) is not a marketed drug, but it is a known CYP inhibitor that is structurally similar to propoxyphene and norpropoxyphene. Propoxyphene, norpropoxyphene, and proadifen were characterized in these studies with CYP3A4(+b5), CYP3A5(+b5) and pooled human liver microsomes. Time-dependent and concentration-dependent loss of activity of CYP3A was measured by formation of testosterone product. Propoxyphene and norpropoxyphene exhibited the greatest inhibition with CYP3A in human liver microsomes, followed by CYP3A4(+b5), and CYP3A5(+b5). Both compounds formed metabolic-inhibitor complexes with vi CYP3A4(+b5) and CYP3A5(+b5), but not with human liver microsomes. Proadifen was a more potent inhibitor of CYP3A4(+b5) than of human liver microsomes and CYP3A5(+b5). The KI values of propoxyphene and CYP3A4(+b5) and human liver microsomes fall within the range of reported therapeutic blood levels of propoxyphene, with reversible inhibition constants (Ki values) above therapeutic blood concentrations for propoxyphene and norpropoxyphene. The KI values of norpropoxyphene and CYP3A4(+b5) and human liver microsomes are higher than most reported blood levels, except for blood levels after repeated dosing of propoxyphene at high concentrations. The predicted change in the area under the plasma concentration versus time curve of an orally administered CYP3A substrate with propoxyphene (AUC'po/AUCpo) was calculated for common CYP3A substrates. The AUC'po/AUCpo ratios are four to twenty-five times higher with co-administration of propoxyphene based on in vitro kinetic parameters. Propoxyphene and norpropoxyphene may cause adverse events when chronically administered at high doses and/or when co-administered with other CYP3A substrates.Item Fatty Acid Synthase, a Novel Target for the Treatment of Drug Resistant Breast Cancers(2009-03-18T18:46:22Z) Liu, Hailan; Zhang, Jian-TingMany cancers, including breast cancer, often develop resistance to chemotherapeutic drugs over a course of treatment. Many factors, including ABC transporter-mediated drug efflux, have been shown to play a role in acquired drug resistance. Fatty acid synthase (FASN), the key enzyme of lipid synthesis pathway, was found to be over-produced in an Adiamycin resistant breast cancer cell line MCF7/AdrVp3000, compared to its parental drug sensitive MCF7 cell line. Inhibition of FASN expression increased the drug sensitivity in breast cancer cells (MCF7/AdrVp3000 and MDA-MB-468), but not in the normal breast epithelia cell line MCF10A1. Enforced overexpression of FASN in MCF7 breast cancer cells decreased its drug sensitivity. These results indicated that FASN overexpression can induce drug resistance in breast cancers. Ectopic overexpression of FASN in MCF7 cells did not affect cell membrane permeability, transporter activity, nor did it affect cell proliferation rate. However, FASN overexpression protects cancer cells from drug-induced apoptosis by decreasing caspase-8 activation. In FASN over-expressing MCF7 cells, I discovered the positive feedback relationship between FASN and activation of Akt as previously reported. However, activation of Akt did not mediate FASN-induced drug resistance. Together with the findings that FASN expression associates with poor prognosis in several types of cancers, my investigations suggest that FASN overexpression is a novel mechanism of drug resistance in breast cancer chemotherapy. Inhibitors of FASN can be used as sensitizing agents in breast cancer chemotherapy.