Developmental toxicology is an important part of preclinical drug toxicology as well as environmental toxicology. Assessing reproductive and developmental toxicity is especially expensive and time demanding, since at least two generations of animals are needed in the tests. In light of this there is a great need for alternative test methods in many areas of developmental toxicity testing.The complete set of RNA transcripts in any given organism is called the transcriptome. Proteomics refers to the study of the proteins in a given organism or cell population. The work of this thesis has focused on the use of high throughput screening methods in transcriptomics and proteomics to search for molecular markers of developmental toxicity.We have studied the global gene expression effects of the developmentally toxic substance valproic acid (VPA) using microarray technology. Several genes were found that display the same gene expression pattern in vivo using mouse embryos as the pattern seen in vitro using the embryocarcinoma cell line P19. Based on these observations, the gene Gja1 was suggested as one potential molecular marker of VPA induced developmental toxicity and potential marker of histone deacetylase (HDAC) inhibition in vitro.Using 2D-DIGE technology, which measures relative protein abundances, the effect of neonatal exposure to the flame retardant PBDE-99 was studied in mouse brain (cortex, hippocampus and striatum) 24 hr after exposure…
Contents
Introduction
Developmental toxicology
Transcriptomics
Proteomics
Toxicogenomics
Biomarkers and “functional biomarkers”
Aims of studies
Methods
Model systems
Valproic acid
PBDE-99
Methods in transcriptomics
Background
Gene expression microarrays
Preprocessing of data
Statistical testing of microarray data
Real time qPCR
Methods in proteomics
Background
Two-dimensional difference in gel electrophoresis (2D-DIGE)
Preprocessing of data
Statistical testing in 2D-DIGE
Protein identifi cation
Gene ontology (GO)
Results and discussion
Transcriptomics
Paper I and II
Experimental design and pooling
h e eff ects of VPA exposure on gene expression in vivo and in vitro
Validation of gene expression results
Current limitations in the use of GO
h e mechanism of VPA induced teratogenicity
Proteomics
Paper III, IV and V
Experimental design
Spot picking
Data analysis
Diff erential expression in predefi ned proteins sets (DEPPS)
Protein isoforms and eff ects of PBDE-99
Conclusions
Concluding remarks and future research
Acknowledgements
References
Author: Kultima, Kim
Source: Uppsala University Library
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