2009 Projects

"Evaluation of the Mechanisms of Nanoparticle Toxicity to Macrophages"
Dr. Andrij Holian

The field of manufactured nanoparticles (particles less than 100 microns in one dimension) has become an intense field of research over the past few years. This is due to the projected high impact that nanoparticles will have in many fields including manufacturing, computers, and human use for imaging and drug delivery. However, while the manufacture of nanomaterials has progressed rapidly, little is known about potential adverse human health impacts of these particles as recently reported by the National Academy of Sciences. Nanoparticles have a number of unusual properties that are due to their composition and extremely small size. Current strategies are to evaluate what properties of nanoparticles can make them more biocompatible.

Our laboratory is currently investigating two different classes of nanoparticles. One group are commercial carbon nanoparticles including C60 carbon spheres, single wall nanotubes and multiwall nanotubes. A number of studies have demonstrated that the carbon nanotubes lead to formation of lung granulomas and that the multiwall tubes can have health effects similar to asbestos fibers. The second group of nanoparticles are titanium dioxide particles produced by a collaborator including nanospheres and nanowires. The titanium dioxide nanospheres are relatively benign, while the nanowires are highly toxic to macrophages and leads to chronic lung inflammation. Currently, our laboratory is investigating the mechanisms of action for both types of nanoparticles. We propose that both types disrupt macrophage membranes leading to macrophage death (called apoptosis).

These studies will use human monocyte derived macrophages, macrophage cell lines, cell culture, enzyme assays and fluorescent techniques to study the mechanisms of nanoparticle action on macrophages. The student will learn how to do sterile cell culture, conduct different enzymatic assays and learn how to use a fluorimeter, confocal microscope and flow cytometery for the fluorescence assays.

"Personalized Medicine, making it a reality for all populations: American Indians and Cancer Therapy. In the Labs of Howard Beall and Mark Pershouse"
Drs. Mark Pershouse and Howard Beall

Pharmacogenomics can be defined as the study of genetic factors that determine if a patient is likely to benefit from, or be adversely affected by, a particular medication. A great deal has been learned from studies of polymorphisms in a small set of genes that determine or predict the outcome of frontline cancer therapeutics. These polymorphisms confer differential responses in patients through altered forms of key drug-metabolizing enzymes, drug transporters or drug targets (Bosch et al., 2006). Knowledge of population frequencies of specific gene polymorphisms (i.e. variants) associated with having an adverse drug reaction is typically based on studies in predominantly non-American Indian populations.

A number of population genetic factors, such as admixture, genetic drift and migration, can skew the distribution of allele frequencies in any one ethnic or cultural group, causing a skewed proportion of individuals with the risk genotype. Our overall hypothesis is that allele frequencies for key gene polymorphisms in understudied American Indian populations are distinctly different from those in previously studied groups. In other words, American Indians can be at higher risk of having an adverse drug reaction, or conversely, they can be at much lower risk.

Allele frequencies for functional polymorphisms in drug metabolizing genes have not been studied in American Indian population for a variety of reasons. Yet this population has a spectrum of cancer incidence similar to other ethnic groups. Our studies attempt to measure for the first time the allele frequencies for several key gene polymorphisms to predict whether our tribal population (Confederated Salish-Kootenai Tribes) is at higher or lower risk for adverse reactions to platinum compounds or quinones.

A summer project in our labs would involve genotyping 50-100 American Indian DNA samples for specific polymorphisms in the XPD1 or NQO2 genes. A student would be taught the basics of DNA isolation, characterization of the purity and yield of these isolations, polymerase chain reaction, operation of automated fragment analyzers such as the Bioanalyzer 2100 from Agilent. The student would also be introduced to basic genetic concepts; Hardy-Weinburg equilibrium, the concept of race at the genetic level, inheritance patterns, sensitivity to privacy issues, and specificity, sensitivity of genetic data.

"The Effects of Woodsmoke Particles on Macrophage Function"
Drs. Tony Ward and Chris Migliaccio

Exposure to biomass smoke can be from either periodic or chronic exposures. Periodic exposures are typified by communities in proximity to forest fires, while chronic exposures are typified by home heating/cooking in developing countries where open fires are commonly utilized. Epidemiological data has shown a link between exposures to biomass smoke and increased incidence in respiratory infections. The main immune cell of the alveolar spaces that is key to responses to inhaled particles is the alveolar macrophage. The present study proposes to assess changes to macrophages following exposure to woodsmoke particles generated from EPA certified woodstoves. The results of these studies will elucidate the mechanism of an increased susceptibility to respiratory infection following exposure to woodsmoke.

In this project, the student will learn animal exposure models, flow cytometry, and sterile techniques, including tissue culture of primary cells.

"Neurotoxin Transport in the Susceptibility to Parkinson’s Disease"
Dr. Erica Woodahl

P-glycoprotein (P-gp) is an ATP-dependent efflux transporter that is important in mediating exposure to drugs and other xenobiotics, such as environmental toxins. P-gp is highly expressed in tissues important in xenobiotic disposition including the blood-brain-barrier (BBB). P-gp transport at the BBB protects the brain from potentially damaging substances circulating in the blood by effluxing them out of the brain. P-gp transport may play a role in Parkinson’s disease by effluxing environmental neurotoxins from the brain and limiting their brain accumulation. Several neurotoxins are associated with the development of Parkinson’s disease including MPTP, paraquat, rotenone, and 6-hydroxydopamine; however, it is not know whether these substances are P-gp substrates.

The ABCB1 gene encodes P-gp and it has been shown that ABCB1 pharmacogenomics – the relationship between an individual’s genotype and drug response – can have a large impact on drug efficacy and toxicity. We hypothesize that ABCB1 pharmacogenomics may also be relevant in neurotoxin transport and the susceptibility to Parkinson’s disease. Changes in P-gp transport, due to ABCB1 genetic variation, may alter neurotoxin brain accumulation and exposure. The main goals of this project are to measure P-gp transport of neurotoxins associated with Parkinson’s disease and estimate the impact of ABCB1 genetic variation on the observed transport.

In this project the student will use an epithelial cell system that expresses either wild-type ABCB1 or an ABCB1 genetic variant. Transepithelial permeability studies will be used to measure neurotoxin transport in ABCB1 recombinant cells. This in vitro system provides the tool to study how xenobiotics move across a physiologic cell barrier, such as the BBB; and therefore, can predict the distribution and accumulation of neurotoxins in the brain. The student will maintain cell culture, quantitate neurotoxins in cells and fluids, and estimate kinetic parameters of neurotoxin transport.
 

"Transport of immunoglobulin A (IgA) across airway epithelial cells in response to environmental pollutants and respiratory infections"
Drs. Zeina Jaffar and Kevan Roberts

The adult human bronchial tree is covered with a continuous layer of epithelial cells that form the conduit for air and are also central players in maintaining defense of the lung against inhaled environmental pollutants and pathogens. The epithelium of the respiratory tract is persistently exposed to a myriad of airborne antigens and must therefore be poised to prevent epithelial colonization by infectious agents. Mucosal surfaces are protected by a first-line defense mediated by secretory IgA, which is composed of two IgA molecules associated with additional peptides, the J chain and secretory component. Epithelial cells play a critical role in maintaining IgA levels in the airway since they express the polymeric immunoglobulin receptor (pIgR) basolaterally that serves to facilitate the transcytosis of dimeric IgA and IgM to the apical surface. IgA associated with pIgR is thought to neutralize pathogens within intracellular vesicular compartments of epithelial cells, while free secretory component has innate antimicrobial properties against a range of pathogens including S. pneumoniae. The pIgR is an integral component of airway and intestinal mucosal immunity, and normally its expression is restricted to mucosal and glandular epithelia, and in hepatocytes in some rodent species. In the airways of mice, pIgR expression is typically low, however, it can rapidly be upregulated by microbial stimulation. The purpose of this project will be to determine the range of environmental agents and cytokines that promote pIgR expression by airway epithelial cell lines. Assay systems monitoring the transcytosis of polymeric immunoglobulins across the epithelium will be developed.

These studies will use epithelial cell lines and the student will learn cell culture, ELISA assays, and fluorescent imaging techniques to evaluate cell expression profiles.

"Efficacy and Safety Assessment for Novel Antitumor Drugs"
Dr. Howard Beall

NAD(P)H:quinone oxidoreductase (NQO1), also known as DT-diaphorase, is a key enzyme in the activation of a class of antitumor agents known as bioreductive antitumor quinones. Interest in this enzyme was prompted by findings that NQO1 was markedly overexpressed in solid tumors from various tissues. This led to speculation that antitumor quinones that are bioactivated by NQO1 may be selectively toxic to those tumors. We have studied a range of benzo-, indole- and quinolinequinones as potential NQO1-directed anticancer agents and identified a number of promising compounds.

An important aspect of drug discovery research is determination of a safety profile for agents with clinical potential. Using previous structure-activity data and a computational approach, we have identified novel quinone structures for development as clinical antitumor agents. Synthesis and characterization of these compounds is currently underway. The plan for this project is determine the toxicity of these compounds to cancer cell lines with and without NQO1 expression. In addition, we will determine their toxicity to cells that are not cancerous, or so called “normal” cells, thereby providing an early indication of the potential safety of these compounds. Further, we will conduct metabolism studies to determine if the novel compounds are efficient substrates for NQO1.

For this project, the student will learn how to culture cells using aseptic technique, how to carry out growth inhibition and cytotoxicity studies, and how to conduct basic studies on the kinetics of enzymatic metabolism.

"Role of adenosine in silica induced inflammation"
Dr. Celine Beamer

Prolonged exposure to crystalline silica (SiO2) in occupational and environmental settings induces chronic lung inflammation which may progress to fibrosis, i.e. silicosis. Despite existing standards in the workplace, silicosis remains a prevalent health problem throughout the world, particularly in developing nations. Insufficient information about the pathophysiological mechanisms that drive silicosis has severely limited the development of effective therapeutic strategies.

Research suggests that the purine nucleotide adenosine may play a role in the initiation, progression and control of pathogenic disorders that involve chronic inflammation and remodeling in the lung. Adenosine is typically present at low levels in human tissues; however in response to stressors, a rapid increase in adenosine levels takes place. Extracellular adenosine represents an alarm molecule that indicates tissue injury, and represents the body’s attempt to re-establish homeostasis through regulation of inflammation. Once generated, adenosine elicits its biological responses through interactions with the four known adenosine receptors: A1, A2a, A2b, and A3. Furthermore, adenosine levels are maintained through the actions of enzyme such as adenosine deaminase. Promising therapeutic approaches to modulate adenosine in health and disease might involve pharmacological compounds that interfere with the breakdown and generation of adenosine, as well as selective agonists and antagonists of the adenosine receptors.

We hypothesize that adenosine may play an important role in the pathogenesis of silicosis through altered levels of adenosine, as well as down-regulation of both adenosine receptors (A2a) and metabolic enzymes (adenosine deaminase). Therefore, in this project, the STEER student will examine lavage fluid collected from saline or silica exposed mice for adenosine levels. In addition, the student will establish the expression pattern of adenosine receptors and metabolic enzymes in tissue sections prepared from the lungs of saline and silica exposed mice. Using this knowledge, the student will design a therapeutic intervention to manipulate an identified adenosine target using pharmacological tools in a mouse model. The student will learn techniques such as HPLC, immunoblotting, immunocytochemistry, fluorescence microscopy, and flow cytometry.