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Johns Hopkins Bloomberg School of Public HealthCAAT

CAAT Grants Program

Research Grants 2011-2012: Summaries

  • Francesca Levi-Schaffer
    Human Culture Model as a Replacement to the Animal Assays for Assessing the Potential of Cosmetic Ingredients to Cause Non-Immunological Contact Urticaria (NICU)
  • Yuhei Nishimura, MD, PhD
    Development of Zebrafish-based Assays for the Assessment of DNT of Chemicals at Low Doses
  • W. Matthew Petroll
    A Novel Anterior Corneal Construct for Ocular Toxicity Testing In Vitro
  • Greet Schoeters
    Biological Pathway Analysis in Human Dendritic Cells after Exposure to Sensitizing Chemicals
  • Thomas Sutter
    A Microarray Analysis Software Package for Comparisons and Extrapolations Relevant to Toxicology
  • Tetsuya S. Tanaka, PhD
    Human Embryonic Stem Cell Lines Amenable to Real-Time Analysis of Transcriptional Activation
  • Hao Zhu, PhD
    Use of Hybrid Modeling Approaches to Develop External Predictive Computational Models for Hepatotoxicity
  • Marie-Gabrielle Zurich
    Brain Aggregate Cell Cultures as an In Vitro Model for Developmental Neurotoxicity Testing

Abstracts

Francesca Levi-Schaffer
The Hebrew University of Jerusalem
Human Culture Model as a Replacement to the Animal Assays for Assessing the Potential of Cosmetic Ingredients to Cause Non-Immunological Contact Urticaria (NICU)

It is well known that a growing number of ingredients present in fragrance mix, nail polish, hair dyes and emulsions can trigger skin reaction recognized by the medical community as contact urticaria. This reaction which is characterized by the appearance of wheal and flare in healthy skin within 30-60 min of the cosmetic product's application leads to significant discomfort. This can happen in both allergic individuals (previously exposed to the same cosmetic product) and in non allergic individuals (who have applied the product for the first time). We have hypothesized that this second reaction, also called non immunologic contact urticaria (NICU), is driven by a direct activation of mast cells, that are cells that reside in the skin. These cells release histamine and other proinflammatory mediators.

Our aim is to develop a reliable replacement of the current animal ear swelling method consisting only of human cells. During the first year of research we developed a co-culture system consisting of human mast cells and skin fibroblasts. We have also assessed using a known mast cell activator the best conditions of mast cell culture and activation (when to measure the released mediator). During the next year of research we will further define the co-culture system, and we will measure mast cell activation/urticariogenic properties of different cosmetic ingredients.

Yuhei Nishimura, MD, PhD
Department of Pharmacology, Mie University Graduate School of Medicine
Development of Zebrafish-based Assays for the Assessment of DNT of Chemicals at Low Doses

Our goal is to develop zebrafish-based assays that can be used to reliably detect developmental neurotoxicity (DNT) caused by exposure to chemicals at low doses. The developing brain is more sensitive to chemical toxicants than the adult brain and exposure during development has been implicated in neuropsychiatric and neurological diseases. The impairments observed in these disorders represent a continuum, from typical clinical syndromes at one extreme to small subclinical deficits in sensory, motor, and behavioral impairment at the other. However, it is often difficult to assess the subtle effects caused by exposure to low concentrations of chemicals that do not induce any external malformation. Large studies in which specific functional domains are assessed using objective instruments are required to detect these subtle changes. The use of non-mammalian animal models allows for testing large numbers of individuals while reducing expense and using fewer mammals. Zebrafish offer several advantages for DNT testing, including the low cost of husbandry, high fecundity, and rapid ex utero development. Furthermore, zebrafish are transparent, allowing for easy observation of morphological changes in the nervous system. Zebrafish larvae also exhibit a number of simple and complex behavioral programs. We propose to develop novel assays to quantify the morphological and behavioral changes in zebrafish following exposure to chemicals at low doses. The assays developed in this study can be performed in a relatively high-throughput manner, making it possible to undertake a systematic and objective analysis and to increase statistical power to detect subtle but significant levels of DNT.

W. Matthew Petroll
A Novel Anterior Corneal Construct for Ocular Toxicity Testing In Vitro

Products to which human eyes may be exposed must be tested to establish their potential to cause ocular irritation.  In vivo ocular irritation tests provide useful information, but can produce variable responses and require the use of animals such as rabbits.  Because corneal damage is the most important factor in determining overall ocular toxicity, an in vitro model of the cornea would have great potential as an alternative to animal testing. A key element to successful tissue engineering of a realistic corneal construct is the development of a 3-D extracellular matrix (artifical stroma) of similar thickness and collagen density to the in vivo tissue, which also supports maintenance of normal stromal cells (corneal keratocytes).  In addition, such a tissue should support the differentiation of corneal epithelial cells, which make up the front surface of the cornea and are thus the first cells exposed to ocular irritants.

We have developed a model in which cell-seeded 3-D collagen matrices are compressed to achieve high stiffness stromal tissue equivalents that support differentiation of corneal keratocytes. We also have developed a unique human telomerase-immortalized corneal epithelial cell line (hTCEpi), which expresses key differentiation markers under stratified, air-lifted culture conditions. Our pilot data suggest that compressed collagen matrices support the differentiation and stratification of hTCEpi under serum-free air-lifted conditions in vitro. The purpose of the proposed research is to further develop and characterize these epithelial-stromal constructs, and to investigate the feasibility of using them as a novel platform for ocular toxicity testing in vitro.

Greet Schoeters
Biological Pathway Analysis in Human Dendritic Cells after Exposure to Sensitizing Chemicals

Skin sensitization caused by low molecular weight chemicals is a major health problem in the industrialized society. Toxicity testing is needed for classifying and labeling chemicals as (non-)sensitizers and for characterizing the associated risk. However, up to now, only animal toxicity studies have been validated for this endpoint and regulatory authorities urge the development of alternative test systems.

In evoking the allergic cascade that is caused by skin allergens, a central role is put aside for dendritic cells (DC) of the skin. In previous studies, an in vitro model for skin DC was developed based on stem cells isolated from human cord blood. Studies on sensitizer-induced gene expression changes in this cell model resulted in a prediction model that successfully classifies chemicals as sensitizers and non-sensitizers, this model has been named VITOSENS. Furthermore, it was demonstrated that the response of this classification model correlates with in vivo predictions concerning sensitizing potency of the chemicals.

The scope of this project is to further elaborate on the signals that rank chemicals according to their sensitizing potency. The underlying hypothesis is based on the danger hypothesis and states that besides the sensitizer-specific antigen signal, cellular damage could function as a necessary trigger to elicit sensitization. Dose-response curves for each signal will be constructed. Insight in the relative contribution of these different parameters would allow 1) the identification of a threshold for chemicals to induce skin allergy 2) more stringent classification of chemicals according to sensitizing potency depending on the strength of the in vitro responses, 3) to understand the differences between skin sensitizers and other compounds that show reactivity towards the skin, such as oxidative stressors and irritants.

Together, the proposed study will significantly contribute to the chemical safety assessment of skin sensitizers making use of 21st century approaches for toxicity testing.

Thomas Sutter
University of Memphis
A Microarray Analysis Software Package for Comparisons and Extrapolations Relevant to Toxicology

Analysis and interpretation of structure activity and dose response relationships, and extrapolations between species and from the in vitro to in vivo situation continue to be major challenges in toxicological sciences.  Establishing mechanism of action and identifying the extent of conservation of mechanism across comparisons is essential to the development of in vitro assays that are predictive of human health consequences.  One promising tool for establishing such relationships is high throughput microarray analysis of mRNA expression.  While proteomic and metabolomic approaches are in development, microarray analysis remains the current standard for high throughput implementation.  Despite the high throughput capacity of microarray, difficulties in analysis including data reduction and visualization (informative clustering) continue to limit the interpretation of such data.  We have developed a multiple comparisons combinatorial analysis approach to the problem of structure activity relationships and have refined the statistical analysis of such data for structure activity relationships and mode of action studies.  Here, we propose to develop prototype software to make these methods available and easily implemented for the toxicological comparisons described above.  Such software is necessary for the important mission of CAAT in the reduction, refinement and replacement of animal use in toxicology.  Specifically, in years 1 and 2, we have developed software that makes available to other researchers our published approach for the analysis of structure activity data.  We have demonstrated that this approach to microarray data analysis is able to: 1) identify common pharmacodynamic action of a series of compounds, 2) provide a rank order of efficacies of these compounds, and 3) identify unique gene clusters indicative of novel pharmacodynamic action.  This later ability distinguishes our approach from more traditional approaches such as hierarchical clustering and principle component analysis.  In this year 3 project, we will improve the current software to make the clustering method more accessible to researchers using the program. Specifically, we will provide improved explanations of the functions of the software.  Furthermore, we will implement tutorials that aid the user in understanding and interpreting the output of the analysis.  Finally, we will link the output clusters to further tools for functional analyses that will aid the investigator in exploring the functional aspects of the gene clusters.


Tetsuya S. Tanaka, PhD
University of Illinois at Urbana-Champaign
Human Embryonic Stem Cell Lines Amenable to Real-Time Analysis of Transcriptional Activation

Human embryonic stem cells (hESCs) offer a unique in vitro platform that can recapitulate cell differentiation events happening in developing human embryos. Because these events are orchestrated by gene expression, one should be able to predict abnormal development of human embryos by observing perturbed gene expression in hESCs as a consequence of administering toxic chemicals. Our objective in this project is to establish hESC lines harboring the fluorescent protein marker that monitors transcriptional activities of genes involved in specification of the neural epithelium. Because gene expression can be observed in real-time in these ESC lines, they will enable simultaneous recording of cellular morphological changes associated with the gene expression. Successful completion of this project will establish about one hundred hESC lines that enable us to monitor unique gene expression during specification of not only the neural lineage, but also other cell lineages. Thus, successful completion of this proposal will significantly reduce use of live animals for broader toxicological studies. Importantly, because neural epithelial cells can be maintained more easily than ESCs, established hESC lines can be distributed as neural epithelial/ precursor cells to the community. Thus, we expect that successful completion of our study will help establish reliable in vitro assay systems for developmental neurotoxicological studies nationwide, which will further reduce use of live animals. Finally, generated hESC lines can be genetically modified further to establish null-mutants or other gene induction, lineage tracing, or marker systems in the future.

Hao Zhu, PhD
Eshelman School of Pharmacy, University of North Carolina at Chapel Hill
Use of Hybrid Modeling Approaches to Develop External Predictive Computational Models for Hepatotoxicity

The objective of this proposal is to develop novel modeling techniques, which can integrate high-throughput chem-informatics and bio-informatics data, to develop robust computational predictors of chemical hepatotoxicity in animals. In our previous studies, we have combined the biological screening data obtained from the HTS bioassays and chemical descriptors to develop statistically significant and externally predictive Quantitative Structure-Activity Relationship (QSAR) models of in vivo toxicity endpoints (including carcinogenicity, acute toxicity and reproductive toxicity). We believe the same or similar approach could be used to study hepatotoxicity and develop the associated toxicity models. In this project, we will first collect data from literature and digital sources and employ special procedures for biological data noise reduction.  Then, building upon successful preliminary results, we will develop rigorously validated and externally predictive QSAR models relying both on chemical and hybrid, chemical-biological descriptors that integrate in vitro assay data with chemical descriptors. Finally, we will deliver a specialized chemical toxicity module, including the toxicity models of this and previous projects, as part of public ChemBench web portal (chembench.mml.unc.edu), providing access both to the curated and harmonized chemical hepatotoxicity database, validated animal hepatotoxicity models, and underlying modeling software. This project will provide the experimental toxicology community and regulatory agencies with robust, animal liver toxicity predictors for environmental chemicals.  Furthermore, we expect the special focus on quality assurance in every step of this project will result in highly significant models that will be ultimately used for reliable regulatory decision support in chemical safety assessment.                         

Marie-Gabrielle Zurich
Brain Aggregate Cell Cultures as an In Vitro Model for Developmental Neurotoxicity Testing

Each brain cell type constitutes a potential target for xenobiotics. Interactions between the different types of cells may also be affected by xenobiotics. A suitable in vitro model for neurotoxicity testing should therefore contain all types of brain cells, and allow the multiple cell-cell interactions involved in brain physiology. Furthermore, for developmental neurotoxicity testing (DNT) it is crucial to use in vitro models that reproduce the critical maturational events allowing cells to reach their final level of differentiation. These characteristics are typical for aggregating brain cell cultures. The cells prepared from rat embryonic brain reaggregate spontaneously into even-sized spheroids, kept in suspension by continued gyratory agitation. All the brain cell types are found in the aggregates, where they interact by physical contacts and by exchange of soluble factors. The maturation process takes about one month. As in the adult brain, a discrete population of undifferentiated stem/precursor cells persists besides the highly differentiated cells in the mature aggregates. The interest of using this model for DNT has been revealed by previous studies. However, in depth characterization is required. The work of the first year will focus on signaling pathways acting on neural stem and progenitor cells. Perturbation of these pathways by ochratoxin A, a developmental toxicant, will be studied. Particular attention will be given to the population of immature cells which persist in highly differentiated cultures. This project will enable the detection of relevant endpoints to monitor the effects of toxicants on critical developmental events, such as proliferation and early differentiation.