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Johns Hopkins Bloomberg School of Public HealthCAAT

Research Grants 2001-2002

Summary of Research Grants

Sensory Irritant Screening Using a Stable Cell Line Expressing the Vanilloid Receptor
Thomas K. Baumann, PhD
Oregon Health Sciences, Portland, Oregon
The recently cloned vanilloid receptor (VR1) has allowed us to proceed with the development of a useful in vitro assay for chemical sensory irritation. This assay will help minimize the use of animals in screening for sensory irritation by chemical agents.

We established a Chinese hamster ovary (CHO) cell line that stably expresses the VR1 vanilloid receptor/ion channel and then tested the effects of various chemicals on the cell line. The VR1-expressing cell line showed membrane currents in response to the sensory irritant piperine (10 µM) and the inflammatory mediator 12-(S)- hydroperoxy-eicosatetraenoic acid (10 µM). Some sensory irritants (e.g. nicotine) were ineffective.

Since the VR1 receptor gates a non-selective cation channel that is highly permeable to calcium, we also measured changes in cytosolic levels of calcium resulting from sensory irritant stimulation. Capsaicin (100 and 311 nM) caused huge increases in the intracellular calcium, as indicated by the ratio of emitted fluorescence intensities for the 340 and 380 nm excitation wavelengths. We also demonstrated that the stable cell line responds reliably with a [Ca2+]i increase in the presence of eugenol (1 mM). Responses to menthol (1 mM) were more variable.

None of the currently available alternative tests is able to assess the sensory aspects of exposure to a chemical. The initial results of this study demonstrate that an in vitro assay can provide a means for assessing the presence of sensory irritants that may excite chemonociceptive trigeminal ganglion neurons and thus give rise to sensory irritation (stinging or burning sensations). The initial success of this approach is encouraging. However, lack of response of the VR1 cell line to some known sensory irritants suggests that a battery of cell lines expressing different irritant receptors may be needed for sensory irritant screening in vitro.
Development of Enzymatic Inhibition Bioelectrodes for the Direct Determination of Phycotoxins
Francesco Botré, PhD
Dipartimento di Controllo e Gestione Delle Merci del Loro Impatto Sull'Ambiente, Universittá Degli Studi Di Roma, Rome, Italy
The goal of this research program is to develop biosensor-based analytical methods for the analysis of phycotoxins (toxins produced by algae which are responsible, when ingested, of various diseases in man) in mussels. These new methods should at least integrate, but hopefully substitute, the commonly applied "global toxicity tests", i.e. highly invasive bioassays on mammalians (in which the mussel extract is either injected or mixed with the food), which are, at present, the only internationally recognized methods for the screening of these toxins in seafood.

Enzymatic inhibition biosensors (ElBs), being a particular form of response-based analytical devices, would reveal a good compromise between purely "biological" and "physico-chemical" techniques. An EIB represents in fact a sort of "artificial bioindicator", partially combining the flexibility of a biological effect-based test and the selectivity of a physico-chernical assay. In other words, the screening analysis to detect the possible contamination of seafood by phycotoxins would be carried out by "feeding the biosensor" and monitoring the alteration of a specific biochemical parameter, rather than "feeding the mouse" and following the development of generic toxic effects.

This second year of the project is aimed to improve the analytical performance of the lab- scale bienzymatic prototype already set up for the direct determination of algal toxins belonging to the DSP ("diarrhetic shellfish poisoning") group; at the same time, other biosensors of the same type will be developed, in view of the realization of a monoenzymatic bioelectrode. The immobilization of only one enzyme is indeed simpler and would markedly improve the lifetime of operation and the stability of the sensor, these parameters being critical towards the subsequent realization of disposable biosensors, produced by the technique of screen printing, to be used for timely planned monitoring of risky areas and possibly also for shipboard operations.
Development and Use of an Immortalized Human Goblet Cell Line for the Use in Ocular Toxicity Testing
Darlene Dartt, PhD
The Schepens Eye Research Institute, Harvard University, Boston, Massachusetts
The conjunctival epithelium along with the cornea make up the ocular surface. The conjunctiva has long been used as a component of the Draize test. Mucin secretion from conjunctival tissue has been one of the endpoints measured by this test. The Draize test has been the accepted method by which a variety of agents have been evaluated with respect to their ability to cause irritation and injury to the ocular surface. Unfortunately the inhumane treatment of animals during this test procedure has often been overlooked. To replace the Draize test, many laboratories have used a variety of cell lines and primary cultures of corneal epithelial cells and conjunctival squamous cells. No one, however, has been able to culture conjunctival goblet cells. The long term goal of our proposal is to develop a cell line of human conjunctival goblet cells with an extended life span which will be able to be used for ocular irritancy testing. We hope to accomplish this goal by fulfilling the following specific aims:

  1. Develop a cell line witn an extended life span by telomerase transfection of primary cultures of human goblet cells;
  2. Characterize the new cell line by using established morphological, histochernical, immunocytochemical and biochemical markers known to be associated with goblet cells; and
  3. Determine the effects of neurotransmitters and growth factors on mucin and protein secretion from this cell line and the effect of ocular irritants on these secretions.

The development of a human goblet cell line with an expanded lifespan that secrete mucin and other antibacterial proteins is a useful model that could be potentially use in place of the Draize test.
In Vitro Irritancy Test Using Telomerase Transfected Human Corneal Cells
James V. Jester, PhD
The University of Texas Southwestern, Dallas, Texas
Eye irritation testing is recognized as important in determining the safety of consumer products where manufacture or use may lead to accidental exposure and damage to the eye. Irritancy testing as currently performed, however, requires the use of live animals for which there are no recognized alternative replacement tests. The long-range goal of our work is to first develop and then validate an alternative, replacement test using a human tissue culture model that reconstructs the anterior, exposed portion of the human eye. For the development of a human based tissue culture model we have generated extended-life, telomerase transfected human corneal cells that show growth regulation and differentiation that is similar if not identical to normal human corneal cells. We are currently using these cells to develop cultured human corneal constructs resembling normal human corneas. Over the final year of this project we will detennine whether responses obtained from these cultured human corneal constructs are similar to the responses previously measured in live animal irritancy tests. These studies should help in establishing an alternative culture model for the elimination of animals in irritancy testing.
Rhythmically Stretching Dynamic Cell Culture: An in vitro Model to Study Particle Cell Injury
Akira, Tsuda, PhD
Harvard School of Public Health, Boston, Massachusetts
Exposure to airborne particulates is strongly associated with lung injury, but the molecular and cellular mechanisms responsible for this association are not fully understood. In this project, we are investigating a new aspect of particle-induced lung pathogenesis: the effect of the physical insults exerted by particles on alveolar epithelial cells. We hypothesize that these physical stimuli may be greatly enhanced by cyclic motion of the alveolar epithelial cells associated with breathing, and may trigger subsequent cell responses. This hypothesis is tested in vitro in a model employing monolayers of the human alveolar epithelial cell line A549 are rhythmically stretched by a cell stretcher device with physiologically relevant tidal breathing conditions, while these cells are exposed to various test particles, such as asbestos fibers. In this year, we will investigate the effects of cyclic cell stretching on particle internalization by the human alveolar epithelial cells, and characterize the overall gene expression profiles of the alveolar cells exposed to particles while the cells are rhythmically stretched by using a state-of-the art DNA microarray technology. The role of receptor-mediated interactions between protein-coated particles and rhythmically moving cell surface receptors (e.g., integrin) will also be assessed. The results of this study would help us to understand how physical insults exerted on the expandingand-contracting alveolar epithelial cell surface lead to lung injury.
Development of an in vitro Cell Model of the Human Blood Brain Barrier
Senén Vilaró, PhD
School of Biology, University of Barcelona, Barcelona, Spain
The development of efficient therapeutic drugs not only requires the optimization of specific and potent pharmacological activity at the target site, but also the efficient delivery of the drug to this site. The main obstacle for efficient drug targeting to the brain lies on the blood-brain barrier (BBB). Separating brain from blood at level of the endothelial cells from cerebral capillaries, this cell barrier strictly controls the concentration and entry of solutes into the central nervous system. To assess the potential toxicity or bioavailability of new drugs in development, academic groups and pharmaceutical companies would benefit from an in vitro model, based on brain endothelial cells in culture, able to mimic the human in vivo BBB. In response to this urgent need and as an alternative method to animal testing, the aim of our proposal is to produce stable, immortalized lines of human brain endothelial cells, which will retain their functional properties, in order to further develop an in vitro cell model of the human BBB for pharmacological and toxicological testing of compounds. Based on the co-culture of astrocytes and immortalized endothelial cells from human brain microvessels, this in vitro cell model will be fully characterized by testing the endothelial cell response to a battery of drugs with known BBB profiles. We are confident that it will represent a valuable alternative to reduce, or even replace, the use of large numbers of animals in drug screening, and will significantly accelerate the CNS-oriented drug discovery and development processes.
Development of Reversibly Transformed Human Corneal Epithelial Cells as an Optimal In Vitro Model
Steven E. Wilson, M.S., MD
University of Washington, Seattle, Washington
For molecular and cellular investigations, as well as toxicity testing, it is frequently useful to have available large quantities of normal human corneal epithelial cells. Unfortunately, sufficient tissue to grow large quantities of these cells are not available. In addition, the expense involved in generating large numbers of primary cells, even if tissue were available, is prohibitive. In the past, several epithelial cell lines have been produced, both in our laboratory and the labs of others, by expressing oncogenes such as SV40 large T antigen, or E6/E7. Unfortunately, all of these cell strains formed, to date, have constitutively (constantly) expressed these oncogenes. Since these oncogenes influence the function of several proteins involved in terminal differentiation and performance of normal functions, for many applications these cells have not been optimal. Our goal in this study is to use a retroviral vector system to transduce E6/E7 into normal human corneal epithelial cells using a vector that can be regulated through the presence or absence of a non-toxic chemical called doxycycline.

We have been successful in generating several strains of these human corneal epithelial cells. We have demonstrated the expression of E6/E7 occurs in the presence of doxycycline, but not in its absence.

We are in the process of evaluating additional epithelial cell functions in these cell lines, including transepithelial permeability to fluorescein and transepithelial electrical resistance in the presence or absence of doxycycline. Epithelial barrier function is an important property of normal corneal epithelium. This is critical in control of corneal hydration, as well as preventing penetration of harmful substances into the cornea. Retention of normal barrier functions by these transduced epithelial cell strains will indicate that these cells are useful for toxicity investigations, as well as cell and molecular studies of epithelial cell function.
Evaluation of CYP3A Induction with Engineered Cell Lines
Bingfang Yan, DVM, PhD
University of Rhode Island, Kingston, Rhode Island
Cytochrome P450 (CYP) enzymes are a family of heme-containing proteins and rank first among the phase I biotransformation enzymes in terms of catalytic versatility and the number of xenobiotics they metabolize. Cytochrome P4503A (CYP3A) enzymes are the most abundant P450 enzymes and involve the metabolism of two thirds of xenobiotics. Numerous drugs and other envirnomental chemicals are known to increase the expression of CYP3A enzymes (induction), thus cause drug-drug interactions. The Food and Drug Administration request all new drugs be tested for CYP induction. Screening of CYP3A induction, however, has been exclusively dependent upon the use of human hepatocytes and animals. The availability of human livers, the use of a large number of animals and potential species difference are the primary limited factors in rapidly predicting the effects of drugs or other chemicals on CYP3A induction. Studies on molecular signaling have revealed that induction of CYP3A enzymes is largely due to transcription activation mediated by the pregnane X receptor (PXR). The proposed studies are designed to develop and validate a PXR-based in vitro system for screening CYP3A induction. The ultimate goal of the proposed studies is to establish stably transfected cells that can be used for screening CYP3A inducers, The transfected cells will express high and stable levels of PXR, and have a reporter gene integrated into the genome. The reporter encodes a luciferase and is subjected to the PXR-mediated regulation upon binding to a CYP3A inducer. Therefore, this system will provide a screening procedure featured by simplicity, high sensitivity and unlimited supplies.
Bovine Corneal Organ Culture: An Ex Vivo Model for Chemical Toxicity Tests
Fu-Shin Yu, PhD
The Schepens Eye Research Institute, Harvard University, Boston, Massachusetts
For five decades, the Draize test has remained the accepted method for evaluating the potential of test material to cause eye irritation or injury. Criticisms of this method center around the inhumane treatment of animals and the irreproducibility of the subjective scoring procedure. There is a great demand for a mechanistic based in vitro testing system that will minimize the use of animals in chemical toxicity tests. Recently, we have adapted a simple, long-term organ culture method as an ex vivo model for chemical toxicity tests. Here, toxicity can be assessed by applying test chemicals to the surface of the cultured corneas. Since this system more closely resembles an in vivo testing system than cell culture, it should serve as an appropriate model for chemical safety tests. The corneas we use are prepared from the bovine (or porcine) eyes, economical and resourceful by-products of meat industry, no live animals are euthanized for testing. Using this system, we will examine the corneal response to chemicals in several paradigms.

  1. The activation of AP- I and NF-kB, two well-known stress-responsive proteins controlling gene expression, in response to chemical stimuli will be used as endpoints for ocular irritancy.
  2. Disruption of corneal barrier function by test chemicals will assess by opacity, epithelial fluorescein retention, and leakage of metabolic enzymes.
  3. Recovery of epithelial function after exposure of the corneal organ culture to tested chemicals will be monitored and used for assigning final ratings of chemicals.

To date, our analyses have demonstrated that corneal organ culture, in combination with the measurements of corneal epithelial permeability and DNA-binding activity of stress-response transcription factors, presents a suitable model for evaluating ocular irritation potential of the innocuous, mild, moderate, and severe irritants. This ex vivo system might be used as an alternative to in vivo Draize test.