NMR-derived developmental metabolic trajectories: an approach for visualizing the toxic actions of trichloroethylene during embryogenesis

Fish embryo toxicity tests for chemical risk assessment have traditionally been based upon non-specific endpoints including morphological abnormalities, hatching success, and mortality. Here we extend the application of H-1 NMR-based metabolomics in environmental toxicology by adding a suite of metabolic endpoints to the Japanese medaka (Oryzias latipes) embryo assay, with the goal to provide more sensitive, specific and unbiased biomarkers of toxicity. Medaka were exposed throughout embryogenesis to five concentrations of trichloroethylene (TCE; 0, 8.76, 21.9, 43.8, 87.6, 175 mg/L) and the relative sensitivities of the traditional and metabolomic endpoints compared. While the no-observable-adverse-effect-level for hatching success, the most sensitive traditional indicator, was 164 mg/L TCE, metabolic perturbations were detected at all exposure concentrations. Principal components analysis (PCA) highlighted a dose-response relationship between the NMR spectra of medaka extracts. In addition, 12 metabolites that exhibited highly significant dose-response relationships were identified, which indicated an energetic cost to TCE exposure. Next, embryos were exposed to 0, 0.88, 8.76 mg/L TCE and sampled on each of the 8 days of development. Projections of 66 two-dimensional J-resolved NMR spectra were obtained, and PCA revealed developmental metabolic trajectories that characterized the basal and TCE-perturbed changes in the entire NMR-visible metabolome throughout embryogenesis. Although no significant increases in mortality, gross deformity or developmental retardation were observed relative to the control group, TCE-induced metabolic perturbations were observed on day 8. In conclusion, these results support the continued development of NMR-based metabolomics as a rapid and reproducible tool for biomarker discovery and environmental risk assessment.