Design and validation of reproductive and multi-generational assays in C. elegant
Approximately 100,000 chemicals are currently in use worldwide with little safety data available for the majority of these chemicals. This is especially problematic in the United States, where more than 60,000 chemicals were grand-fathered in via the 1976 Toxic Substances Control Act. The difficulty in assessing safety of chemicals not only arises from their large number but also from the complexity of biological effects they elicit over a large range of doses. Furthermore, some endpoints are inherently difficult to observe in traditional toxicity tests such as reproductive processes that unfold over many months in mammals as well as epigenetic effects that can span multiple generations. Here, we propose to directly address these issues by developing and adapting a novel assay in a genetic model system, the worm C. elegans. Specifically, we are taking advantage of the evolutionary conservation of epigenetic pathways and of the vast number of genetic tools in C. elegans to detect environmental chemical disruption of the germline chromatin's epigenome. By monitoring over multiple generations changes in epigenetic marks such as histone modifications, we will be able to identify environmental chemicals for their ability to disrupt the epigenome as well as understand the mechanisms of pathway perturbations leading to these changes. Therefore, these studies aim at developing unique methods to interrogate our chemical environment for its effect on germline maintenance and health.
Maria Teresa Cruz
In chemico, in silico and in vitro modeling to predict human respiratory allergens
The prevalence of respiratory allergies has dramatically increased over the last decades due to environmental and occupational factors, being occupational asthma the most prevalent occupational lung disease in developed countries with high levels of morbidity. More than 300 substances have been shown to cause occupational asthma, and a large proportion of these are low molecular weight (LMW) organic compounds. There is currently no widely accepted animal or non-animal method able to identify potential LMW respiratory sensitizers for regulatory purposes, despite the risk to human health. In addition, there is significant social, scientific and economic pressure to replace animal testing where possible. Therefore, development of non-animal assays for identifying potential respiratory allergenic chemicals is highly warranted to protect public health and will be of uttermost importance for the pharmaceutical, chemical, cosmetic, pesticide, and food industries. In this context, and supported by our expertise in the development of predictive in vitro toxicity tests, we intend to develop an innovative platform to predict respiratory sensitization hazard. For that, in chemico measurements of respiratory allergens with model peptides will be performed to assess the reactivity of respiratory allergens. In addition, the adjuvanticity/irritancy and immunogenicity triggered by respiratory allergens will be assessed in cells representative of the respiratory system. Finally, we will design a mathematical framework, derived from the readouts described above for the identification and classification of respiratory sensitizers.
Epithelix Sarl has provided a line of credit to Dr. Cruz for these studies. Epithelix produces standardized in vitro human lung tissue. We offer our sincere appreciation to Epithelix for this contribution.
Differentiated human respiratory epithelial cell cultures as a surrogate system for assessing the effects of estrogenic compounds on pulmonary disease pathogenesis
The beneficial and harmful effects of natural estrogen-based therapies, alternative plant-derived estrogen therapies, and inadvertent exposure to xenoestrogens in the environment are debated, with consideration limited to the effects on reproductive tissues and cells. An in vitro system for screening the impact of hormonal and environmental substances on human cells is needed, with attention paid to cells and tissues outside of the reproductive system. The development of pulmonary diseases differs between the sexes and is affected by estrogen exposure. Females tend to suffer more severe pulmonary disease than males, which is partly caused by heightened inflammation. Severe pulmonary inflammation in females can be reversed by treatment with estrogen-a potent anti-inflammatory agent. This proposal will characterize how estrogens affect influenza virus replication, cell function, and inflammation, using an in vitro differentiated human respiratory epithelial cells (hREC). Respiratory epithelial cells are the primary cell type infected with influenza viruses and provide an alternative model for testing the therapeutic or harmful effects of natural estrogens, phytoestrogens (e.g. genistein from soy), and xenoestrogens (e.g. Bisphenol A from plastics) on responses to infection. The long-term goal of this proposal is to replace whole animal testing with an alternative - in vitro hREC cultures. This in vitro system for screening the impact of hormonal and environmental substances on human respiratory epithelial cell function could be expanded to include cellular responses to toxins, allergens, pathogens, and other environmental contaminants.
Epithelix Sarl has provided a line of credit to Dr. Klein for these studies. Epithelix produces standardized in vitro human lung tissue. We offer our sincere appreciation to Epithelix for this contribution.
Novel axial elongation morphogenesis systems using embryonic stem cells to investigate teratogenic factors
The objective of our research is to generate culture systems for mouse and human embryonic stem cells, which can be used to identify drugs and gene mutations that potentially induce birth defects in human. We have established culture systems using mouse cells, which display a highly unique morphological change, i.e., transformation from spherical to elongated cell aggregates. We have shown that this elongation morphogenesis has molecular characteristics similar to the cranial-caudal, or head-to-toe, body axis of normal embryos. Thus, our culture system can mimic the body elongation process that normally occurs in the embryo during early gestation period. Disturbance in axial elongation morphogenesis could cause severe birth defects, such as caudal dysgenesis and neural tube closure defects. However, their etiology and pathological mechanisms are still not well understood. Most studies on birth defects have been conducted using model mammalian species, such as mice and rats, in which many animals are experimented and sacrificed. However, once we demonstrate that our culture systems can serve as valuable tools to investigate the mechanisms of birth defects, the use of model animals for birth defect research should be substantially minimized. The proposed project focuses on molecular profiling of our elongation systems to obtain information on what kind of genes are involved in this specific morphogenesis. We are also conducting chemical screening to identify drug compounds that interfere with the elongation morphogenesis as substances to cause birth defects, i.e., teratogens.
Nicole zur Nieden
Skeletal teratogenicity of environmental chemicals predicted with human induced pluripotent stem cells in vitro
Birth defects that affect musculoskeletal tissues account for 5% of all infant deaths. They may be caused by chemical by-products released into the environment or chemical ingredients in pesticides, fungicides, and paints imposing substantial burdens on the affected individuals and families. Evaluating the safety of such chemicals using a suitable prenatal model of human embryos is therefore an essential scientific and societal goal. Screening is needed to uncover which chemicals might potentially cause such devastating birth defects. However, traditional in vitro screening tests still require the killing of animals or the in vitro use of the ethically challenged embryonic stem cells. This project aims to provide a reliable in vitro developmental screening assay that will replace traditional animal methods and improve in vitro screening by utilizing differentiating human induced pluripotent stem cells to model the developing skeleton of the embryo. These cells are artificially made from adult cells and reverted back into an unspecialized state from which they may develop into all possible cell types, among them bone-forming cells. With this ethically acceptable model, tolerable exposure levels of environmental chemicals could then be based on the individuals in the population that are most vulnerable, namely the developing embryo.