Generation of biological assays to evaluate the activity of cellular stress pathways as beacons of toxicological effects of dietary additives and contaminants

Línea de investigación

Análisis de la señalización celular durante el desarrollo de epitelios en Drosophila melanogaster.

Descripción

Our group has considerable experience in the use of Drosophila melanogaster as an experimental model to address developmental biology problems (21 years of experience funded by 8 consecutive National Plan projects). Our experience lies in applying genetic and molecular biology techniques to understand gene function during epithelial development. We have used genome-editing techniques, reporter constructs assays and genomic approaches to analyse gene regulation and cell signalling in the Drosophila wing epithelium. We now want to develop novel sensors of cellular distress, and to optimise the use of cell stress response pathways (CSRP) as indicators of adverse effects of food additives and impurities.

General Objective

Develop, validate and implement new approach methodologies to evaluate cellular and organismal adverse effects of emerging food and environmental contaminants. 

Specific Objective

1. Toxicological characterization of Drosophila larval guts exposed to reference toxicants (DNA damage, Oxidative stress and ER stress).
2. Identify cellular stress response pathways (CSRP) downstream genes in Drosophila guts. Transcriptomic signatures serve as first-tier identifiers of chemical adversity and provide a set of biomarkers eligible for toxicity testing and hazard identification. Stress response pathways ultimately result in the transcriptional activation of cytoprotective genes, which are ubiquitous and display broad stressor responses. We expect that monitoring in parallel the activity of several stress pathway-specific genes would allow us to define a complete picture of the cellular status, providing quantitative measures of the effects of toxicants on cellular homeostasis. We will collect Drosophila larval guts for transcriptomic analyses following acute exposure conditions to reference toxicants.
3. Generate and characterize the activation of CSRP reporter constructs in Drosophila. From the set of genes showing maximal expression changes after exposure to reference toxicants in Drosophila guts, we will study: i) participation in different CSRPs, ii) expression of mRNA in larval tissues (mRNA in situ hybridization in gut, fat body, salivary gland and imaginal discs), iii) genomic structure, searching for DNA sequence conservation with other invertebrates in the promoter region (2 to 4 Kb 5´ to the transcription start site) and iv) presence of consensus binding sites for the known transcription factors acting in each CSRP. Drosophila regions of 2-4 Kb in size will be amplified by PCR using genomic DNA as a template, and the PCR products will be subsequently inserted into a GFP expression/integration vector to generate transgenic flies.

Contacto

José F. de Celis Ibeas.

Correo electrónico: jfdecelis@cbm.csic.es.