Graphene nanomaterials have attracted great research interest for their potential applications in electronics, energy, materials and biomedical areas. However, little information is available about their toxicity mechanisms. In this study, the potential hazard of graphene nanomaterials were investigated in the nematode Caenorhabditis elegans, using an integrated systems toxicology approach. To gain an understanding of the underlying molecular mechanisms of graphene toxicity, microarray, metabolomics, and lipidomics assays were performed, followed by an integrative pathway analysis. Results suggested the daf-16
/FOXO pathway, drug metabolism, Fat metabolism, and MAPK pathway as potential toxic mechanisms of GO toxicity. To validate the detected pathways, functional genetic studies were performed, using loss-of-function mutants of genes of those pathways. Involvement of daf-16
/FOXO pathway in neuro-behavioral toxicity was validated by increased nuclear translocalization of the transcription factor daf-16
by graphene exposure. Hence, we provide insights into the mechanism of toxicity of graphene and suggest the direction for the further examination of graphene toxicity. The comprehensive approach allowed us to integrate all assay results in a consistent understanding of graphene toxicity. Thus, this approach will serve as a proof-of-principle for the general suitability of multi-OMICS approaches for the elucidation of toxicity and stress responses, providing valid hypotheses which allowed to obtain unbiased knowledge.