Development of bioassays for herbicide toxicity based on gene expression profiling using yeast cells (HERBITOXBIOAS)

Principal investigator: Cristina Anjinho Viegas

Participating institutions: Instituto Gulbenkian de Ciência, IMAR – UCoimbra

Contract: PTDC/AMB/64230/2006   - Ciências e Tecnologias do Ambiente

Start date: 01/11/2007

Duration: 36 months

 

Pesticides released intentionally or by accident into the Environment may cause hazard to terrestrial and aquatic ecosystems, ultimately to human health. Eco(toxicological) evaluation of new or existing pesticides and the development and validation of bioassays that may be applied in the diagnosis of low levels of these xenobiotics in environmental samples is of major importance. In this context, there is an urgent demand to develop and validate more sensitive and rapid screening tools that may be complementary and/or alternative to whole-organisms toxicity tests and minimizing animal utilization. The ultimate goal of this project is to develop novel sensitive screening tools based on gene expression profiling in the microbial eukaryotic model Saccharomyces cerevisisae that should be valuable for the diagnosis of herbicide toxicity in environmental samples and/or as a preliminary toxicity test. The yeast is non-pathogenic, very easy to maintain and cultivate, with the genome fully disclosed.  Studies on the response to xenobiotic stress in this experimental model system should provide evidences of mechanisms of action and of toxicological indicators that may be potentially meaningful for mammals, aquatic organisms and others, in chemical risk assessment, and used as reference to orientate experiments or toxicity tests in more complex and less experimentally accessible eukaryotes. Changes in gene expression are among the first responses to chemical or environmental stress exposure and may be a very sensitive parameter to detect potential adverse effects of low sub-lethal doses of bioavailable chemicals. Specifically, the project aims primarily to gain information on changes occurring in the yeast transcriptome as a result of cells exposure to sub-lethal and environmentally relevant levels of herbicides from different chemical classes, by using Affymetrix GeneChip Yeast Genome 2.0 arrays (Tasks 1 and 2; collaboration between the teams from Instituto Superior Tecnico - IST, and Instituto Gulbenkian de Ciência - IGC). Whenever adequate, mRNA quantification using Northern blot hybridisations or quantitative two-step RT-PCR may be used to confirm and get a clearer picture of the dose- and/or time-dependency of the expression of particularly relevant genes (in IST). Results to be obtained will enable the identification of genes that are up- or down-regulated in response to each toxicant exposure and the estimation of indices of herbicide toxicity with basis on dose-response data concerning changes of the expression of relevant genes. One main objective of the project is to identify yeast genes of interest whose expression is modified in response specifically to a particular herbicide or a chemical class of herbicides. This information will be valuable to identify yeast genes whose expression could be used as indicators of the potential toxicity of these xenobiotic compounds and to allow the establishment of gene expression signatures enabling discrimination between herbicides. In order to evaluate the suitability of gene expression profiling in yeast as a tool for herbicide (eco)toxicological assessment, the patterns of altered expression of relevant genes and levels of toxicity estimated by this approach will be linked to conventional indices of toxicity obtained using ecologically relevant bioassays, based on experiments using a “run-off” test chamber. This simulator system, developed by the team of Instituto do Mar (IMAR)-Centro Interdisciplinar de Coimbra, mimics as far as possible the application and fate of the herbicides under study in and through an agricultural reference soil allowing the collection of soil, run-off water and leachate samples for further ecotoxicological testing (Task 3; collaboration between the teams from IST IMAR). Additionally, further studies viewing the confirmation and elucidation of the role on herbicide toxicity and response in yeast of particular genes of interest identified based on the global genome analysis from Task 2, will be also carried out (Task 4; in IST). Model herbicides to be examined will be selected among widely characterized plant protection products from different chemical classes and having different modes of action that have been used mostly in diverse crops in the European Union, particularly in Portugal, and whose residues are likely to contaminate surface and ground waters. In conclusion, the herein proposed project will contribute to the development and evaluation of sensitive and relatively easy-to-use yeast-based bioassays to be used as screening tools for herbicide monitoring in environmental samples and/or for the comparative assessment of the potential toxicity of herbicides (presumably as one of a set of toxicity tools integrating responses at different biological levels for use in ecotoxicological risk assessment), with high impact in the field of Environmental Sciences.