Browsing by Subject "Cytotoxicity"
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Item Alteration of immune responses by cocaine: Cytokines and reactive nitrogen intermediates(Texas Tech University, 1998-08) Starnes, Joel D,In recent years cocaine abuse has reached epidemic proportions. The use of cocaine, like many other drugs of abuse, has been shown to have immunomodulatory consequences. However, the effects of cocaine on the immune system have not been thoroughly examined. The macrophage (M0) plays a central role in the regulation of inflammatory responses, antigen presentation, and is an important non-specific first line of defense against incoming pathogens. In the present studies, the immunomodulatory effects of cocaine on certain aspects of M0 functions were examined. Cocaine was administered to experimental mice and/or M0 were exposed to cocaine or one of its metabolites in vitro. Experiments were conducted to determine the effects of cocaine on the ability of M0 to produce cytokines and reactive nitrogen intermediates (RNI). A novel method of cytokine induction using a mannose receptor ligand was also developed. Mannosylated bovine serum albumin (M-BS A) was administered to a variety of cell types including M0, and shown to induce cytokine secretion. M0 from cocaine-injected mice displayed a reduced ability to secrete proinflammatory cytokines when stimulated with M-BSA. This effect was also demonstrated by M0 exposed to cocaine in vitro. However, due to the acute nature of cocaine's effects, it had no effect on the secretion of regulatory cytokines. Cytokine mRNA levels were unaffected by cocaine exposure, suggesting the mechanism of inhibition involved attenuation of cytokine secretion, but not transcription. In vivo cocaine administration, but not in vitro cocaine exposure, caused inhibition of RNI in M0. In vitro exposure to the metabolite norcocaine inhibited nitrite production, suggesting a possible in vivo mechanism. iNOS levels were analyzed via Western blot, showing that iNOS protein levels were unaffected by norcocaine. The mechanism of inhibition appeared to be through the attenuation of iNOS enzymatic activity. Collectively, these studies indicate that cocaine impairs M0 proinflammatory activity and has the ability to cause immune system compromise. These studies suggest that the immunoregulatory effects of cocaine may lead to increased instances of infectious disease in drug addict populations.Item Characterization of VOPQ, A Type III Secreted Effector Protein from Vibrio Parahaemolyticus(2009-06-15) Burdette, Dara Lesley; Orth, KimVibrio parahaemolyticus is a Gram-negative bacterium responsible for gastroenteritis associated with the consumption of raw or undercooked shellfish. Its most well-characterized virulence factors are hemolysins that cause b-hemolysis on a special blood agar. Mutants lacking these hemolysins are still virulent in animal and tissue culture models of infection. These phenomena can be attributed in part to one of two type III secretion systems; one on chromosome 1 and the other on chromosome 2. We demonstrate that Vibrio parahaemolyticus utilizes the type III secretion system on chromosome 1 to induce a temporally regulated series of events that initiates with the induction of autophagy, followed by cellular rounding and finally cellular lysis and death. To the best of our knowledge, no other Gram-negative extracellular bacterium has been shown to induce autophagy during infection. To understand the mechanism of Vibrio parahaemolyticus induced cell death, we focused our analysis on VopQ, a type III secreted effector encoded by the type III locus on chromosome 1. We demonstrate that VopQ contributes to cytotoxicity as DvopQ strains induce cell lysis less efficiently. In addition, VopQ is necessary and sufficient for the induction of autophagy during infection. VopQ-mediated autophagy occurs independently of phosphatidylinositol 3-kinases and prevents phagocytosis. Additional experiments using Saccharomyces cerevisiae demonstrate VopQ induces autophagy and cell death through an evolutionarily conserved mechanism. Results presented herein delineate a novel virulence mechanism used by Vibrio parahaemolyticus to cause disease. This study also highlights the effector VopQ as a novel inducer of autophagy and a key mediator of cytotoxicity during infection.Item Cytotoxicity of Selenocyanobiotin Against MDA-MB-231 Breast Cancer Cells(2008-12) Tsai, I-Huei; Spallholz, Julian E.; Roman-Shriver, Carmen; Boylan, Lee M.Numerous selenium compounds have demonstrated their ability to act as a chemotherapeutic agent through their pro-oxidative activity in cancer therapy in vitro. However, one major problem in cancer therapy is tumor drug specificity. Previous studies have shown that the membrane bound folate receptors (FR) and biotin receptors (BR) are over expressed in numerous cancer cells but not in normal cells. The present study was aimed to determine whether a selenium-biotin conjugate can be internalized via a BR-overexpressing human breast cancer cell line, MDA-MB-231. It was observed that MDA-MB-231 cells treated with 1, 5, 10, and 20 µM of selenocyanobiotin (SeCN-biotin) after 24-, 72-, and 120-hour treatment resulted in inhibition of cell proliferation and cell death as determined by the [methyl-tritium] DNA thymidine incorporation assay. The experimental results provide support for the hypothesis that SeCN-biotin could penetrate into BR-over-expressing cancer cells and inhibit DNA synthesis in a time- and dose-dependent manner in vitro.Item Dendrimers as drug and gene delivery vectors : a self consistent field theory study(2013-08) Lewis, Thomas Wade Stakesby; Ganesan, VenkatThis research focuses on the modeling of dendrimer molecules for their application as delivery vectors within drug and gene therapy systems. We examine how the architecture and composition of dendrimers affect their drug and gene binding efficacies along with their interactions with anionic bilayers. We specifically focus on how the weakly basic nature of dendrimer monomers and the addition of neutral grafts to dendrimer surface groups affect their interactions with drugs, linear polyelectrolytes, and bilayers. By using polymer self-consistent field theory (SCFT) to model such systems, we develop a computationally efficient means to provide physical insights into these systems, which are intended to guide dendrimer design for delivery applications.We study the conformational properties of weakly basic (annealed) polyelectrolyte dendrimers by developing a SCFT model that explicitly accounts for the acid-base equilibrium reaction of the weakly basic monomers. We specifically focus on the role of local counterion concentration upon the charge and conformations of the annealed polyelectrolyte dendrimers. We compare our results to existing polymer scaling theories and develop a strong stretching theory for the dendrimer molecules.We extend the previous study to model the interactions between weakly basic dendrimers and weakly acidic, hydrophobic drug molecules. We specifically examine the effects of excluded volume, electrostatic, and enthalpic interactions on the binding efficacies between dendrimers and drugs under a variety of dendrimer generations, solution pOH conditions, drug sizes, and Bjerrum length values.We study the role of neutral dendrimer grafts on the conformations and drug binding efficacies of dendrimers. We then elucidate how the observed conformational changes affect the charge of the dendrimers. Furthermore, we examine how the presence of grafts affects the steric, electrostatic, and hydrophobic interactions between the drugs and dendrimers under a variety of solution conditions. We compare our results with the binding efficacies observed for non-grafted dendrimers to delineate the conditions under which the grafted dendrimers are better suited as drug hosts.We include semi-flexible, anionic linear polyelectrolyte (LPE) molecules in our grafted dendrimer SCFT framework to model the interactions between dendrimers and negatively charged genetic materials. Specifically, we examine how neutral dendrimer grafts, LPE stiffness, and solution pOH affect the interactions between dendrimers and LPEs. We then use our SCFT potential fields as input into Monte Carlo simulations in order to determine the dendrimer-LPE potentials of mean force and the resulting loop and tail statistics of the dendrimer-adsorbed LPE chains.We incorporate a negatively charged bilayer into our grafted dendrimer SCFT framework to model dendrimer interactions with a cellular membrane. We specifically examine the role of dendrimer grafting length, solution pH, and membrane tension on such interactions. By comparing our results with SCFT calculations of fixed dendrimer conformations and hard sphere nanoparticles in the presence of membranes, we delineate the role of dendrimer flexibility and porosity on the interactions between dendrimers and anionic bilayers.Item In vitro evaluation of the cytotoxic potential of Brickellia cavanillesi (Asteraceae) using HepG2 cells(Texas Tech University, 2007-05) Viñas, René; Smith, Ernest E.; Tang, Lili; Gao, WeiminCharacterization of the potential toxicity and biological mechanisms of alternative herbal medicine offer significant benefits for people using them to treat chronic diseases or symptoms. The Latin American tea "prodigiosa" (Brickellia cavanillesi), a member of the Asteraceae botanical family is traditionally consumed as an herbal remedy to treat diabetes and other chronic diseases in several underdeveloped countries. However, there is limited data on the potential toxicity or mechanism of action for B. cavanillesi. Consequently, in this study we used an in vitro cell culture approach to evaluate cellular toxicity of an aqueous extract of B. cavanillesi. Hepatocellular carcinoma cells (HepG2) were submitted to both a dose- (IC50) and a time-dependent toxicity evaluation in the presence or absence of Fetal Bovine Serum (FBS). Herbal extracts (1-35%) were used for the IC50 study. In the time course study, cells were exposed to 1-10% extract solutions of B. cavanillesi for 48hrs. Cell viability was determined with the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay. No significant differences were observed when cells were cultured with or without FBS. In this study the IC50 ranged from 5-10% in the absence and presence of FBS. At doses exceeding 10%, cellular viability decreased significantly (pItem Isolation and effects of citrus limonoids on cytochrome p450 inhibition, apoptotic induction and cytotoxicity on human cancer cells.(Texas A&M University, 2007-04-25) Poulose, Shibu M.This dissertation illustrates an efficient purification method for citrus limonoids and flavonoids, while examining their effects on cytochrome P450 inhibition and apoptotic induction on human neuroblastoma (SH-SY5Y) and colonic adenocarcinoma (Caco-2) cells. The first study developed a bulk purification method for limonoids, from seeds and molasses of citrus fruits, using a combination of chromatographic techniques. This also resulted in an efficient purification method for naringin and hesperidin from citrus byproducts. The second study investigated the inhibitory effects of purified limonoids and flavonoids on the enzymatic activities of different isoforms of human cytochrome P450. O-Dealkylase and hydroxylase activities of CYP1A2, CYP1B1, CYP3A4 and CYP19, using specific substrates such as ethoxyresorufin (ethoxyresorufin O-dealkylase, EROD), methoxyresorufin (methoxyresorufin O-dealkylase, MROD), and dibenzylfluorescein (DBF), were found to be significantly (P < 0.001) reduced at micromolar levels. A kinetic analysis showed competitive and non-competitive modes of inhibition by limonoids, on CYP19 hydroxylase activity. The results corroborate the active role of limonoids in the redox cycling mechanisms. The third study examined the antioxidant and apoptotic inducing ability of limonoid glucosides on human neuroblastoma cells. Four limonoid glucosides, LG (17beta-D glucopyranoside limonin), OG (obacunone 17beta-D glucopyranoside), NAG (nomilinic acid 17beta-D glucopyranoside), and DNAG (deacetylnomilinic acid 17beta-D glucopyranoside), have shown superoxide scavenging at millimolar levels. Micromolar amounts of LG and OG induced rapid necrosis of SH-SY5Y cells. Cytotoxicity was correlated (P = 0.046) to a concentration and timedependent increase in caspase 3/7 activity. Analyses of DNA content during the S phase of the cell cycle indicated reductions of 86.6% for LG and 82.3% for OG as compared to untreated. The results validate the antineoplastic distinctiveness of limonoid glucosides. In the fourth study, cytotoxic effects of limonoid aglycones and glucosides were assessed on human SH-SY5Y neuroblastoma and colon carcinoma (CaCo-2) cell lines and compared with the non-cancerous Chinese hamster ovary (CHO) cells. Significant (P < 0.001) cytotoxic effects were observed only on cancerous cells, over 24 to 36 h. The study revealed a marked increase in the DNA content of aneuploidic cells, which results in cell cycle arrest. The results confirm that glycosides are the most active apoptotic inducing form.Item Synthesis and evaluation of small molecule DNA-interactive compounds : total synthesis of (±)-NNN-5'-acetate, synthesis of skipped benzimidazolium aza-enediynes, and synthesis of a series of C2-aryl UK-1 analogs(2009-08) Marriner, Gwendolyn Ann; Kerwin, Sean M.; Magnus, Philip D.; Iverson, Brent L.; Brodbelt, Jennifer S.; Whitman, Christian P.Small-molecule DNA interactive compounds are critical as both carcinogens and therapeutic agents. In this research, a synthetic precursor to a known carcinogen, (±)-N’-nitrosonicotine-5’-acetate was synthesized, and its interactions with DNA were evaluated by polyacrylamide gel electrophoresis and electrospray-ionization mass spectrometry. A library of skipped benzimidazolium aza-enediynes which selectively target unmethylated cytosines in presence of unmethylated cytosines were synthesized, and their biological properties were evaluated in a nicking assay and cytotoxicity study. Finally, a series of structural analogs to a antineoplastic agent, UK-1, were synthesized via a biaryl coupling at C2 on the benzoxazole.Item Synthesis, Characterization, and Toxicity Studies of Dirhodium and Diiridium Metal-Metal Bonded Compounds(2012-10-19) Lane, Sarah MargaretThe anticancer properties of dirhodium tetraacetate were discovered in the 1970's, and subsequently motivated the research of several dirhodium paddlewheel derivatives. The promising results of this research led the Dunbar group to investigate the biological properties of dirhodium partial paddlewheel compounds. Previous work in our group has focused on dirhodium carboxylate derivatives with a series of diimine ligands, namely 1,10-phenanthroline (phen), dipyrido[3,2-f:2',3'-h]quinoxaline (dpq), dipyrido[3,2a:2',3'c] phenazine (dppz), and benzo[i]dipyrido[3,2-a:2',3'-c]phenazine) (dppn). Current research has expanded this diimine series by substituting the carobxylate bridging group with p-methoxyphenylphosphine (PMP). This new series of compounds was characterized by several techniques, including: X-Ray crystallography, 1H NMR spectroscopy, and electronic absorption spectroscopy. The cytotoxicity of these compounds towards HeLa cells was investigated in presence and absence of light in an effort to investigate the ability to use these compounds as photodynamic therapy (PDT) agents. Cytotoxicity measurements were carried out using the 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide (MTT) assay. It was found that in the dark [Rh2(PMP)2(dppz)2][BF4]2 (the dppz derivative of the dirhodium PMP compound) had no cytotoxicity towards HeLa cells, but experienced a 7 fold increase in cytotoxicity upon irradiation (with lambdai_rr equal to 350 nm). This dramatic increase in cytotoxicity upon irradiation makes this compound a potential PDT agent. Diiridium (II,II) compounds were prepared in a dual attempt to determine how the properties of the dirhodium core effect the biological activities of these compounds, as well as investigate the biological activity of a set of compounds that has yet to be explored. The compound [Ir2(DTolF)2(CH3CN)6][BF4]2 was chosen because it has a well understood dirhodium analogue, and it is a known compound. However, it was discovered that there was a potential silver contamination in the final product, stemming from the silver trifluoroacetate oxidant used during synthesis. Consequently, a new method of preparing this compound was required. The new synthetic pathway for the diiridium compound [Ir2(DTolF)2(CH3CN)6][BF4]2 was devised, and the cytotoxicity and photocytotoxicity studies were performed for the first time (to our knowledge) on a diiridium (II,II) compound. Despite the stability of the compound, it was determined to be highly toxic, both in the dark and upon irradiation.Item Systematic Approach to Compare the Inflammatory Response of Liver Cell Culture Systems Exposed to Silver, Copper, and Nickel Nanoparticles(2011-10-21) Banerjee, NiveditaAlthough nano-sized metal colloids are used in industrial and medicinal applications, little is known about the potential liver toxicity of these materials after occupational or intentional exposures. To begin to resolve some outstanding hepatotoxicity concerns, the inflammatory response of hepatocytes after exposure to metal colloids was assessed. Four ~30-nm-sized metal colloids, including silver (nano-Ag), copper (nano-Cu) and nickel (nano-Ni) were examined in an effort to understand the induced cytokine expression in a murine liver cell line (AML12). Here we also utilized another system, co-cultures of hepatocytes, Kupffer?s cells, and lymphocytes isolated from C57BL6 mice. Cells were exposed to the materials over dose-response (0.1mg/L to 1000mg/L) and time-dependent (4 h, 48 h, and 1-week) studies. Cytotoxicity was measured via metabolism of resazurin and validated via MTT assay and cell counts. Inflammatory response was determined by cytokine profiles (TNF-a and IL-6), as well as by mRNA and protein expression of heat shock protein (Hsp70). Results from cells exposed to nano-Ag to doses of up to 100mg/L exhibited no significant changes in cytotoxicity, IL-6, or TNF-a production, or Hsp70 expression. Both nano-Cu and nano-Ni exposed cells exhibited decreased metabolism, increased Hsp70 induction, and increased inflammatory responses (IL-6 and TNF-a). Dynamic light scattering and electron microscopy were used to characterize particle size and surface charge. All three metal colloidal systems demonstrated different particle size distributions, agglomerated sizes, and surface zeta potentials. Furthermore, each metal colloid system elicited different inflammatory biomarker responses and stress protein expression.