A novel approach for testing the teratogenic potential of chemicals on the platform of metabolomics: studies employing HR-MAS nuclear magnetic resonance spectroscopy

Abstract

NMR based metabolomics offers a complementary approach that gives information on whole-organism functional integrity over time after drug exposure. Hence the objective was to develop a quick, reliable method for testing the teratogenic potential of a new chemical entity (NCE) on the platform of metabonomics, as an alternative to conventional procedures. Time mated Charles Foster rats (n = 9) were injected with cyclophosphamide (0, 5, 15 and 30 mg kg⁻¹) at different dose levels on day 11 of the pregnancy, through an i.p. route. On day 12 of the pregnancy, embryos were procured from six rats out of the 9 pregnant rats from each group, using a per abdominal approach. These embryos were then undertaken for morphological studies and NMR experiments using a high resolution-magic angle spin (HR-MAS) probe. The remaining three rats were followed until term to procure full term pups, which were used for conventional observations and studies. Multivariate unsupervised principal component analysis of the ¹H NMR spectra from the rat embryos revealed a dose dependent cluster separation between the controls and treated specimens, which was further confirmed by supervised partial least-squares discriminant analysis with an R² of 0.77 and Q² of 0.72. Those embryos which were found to have malformations/anomalies, significantly presented a few upregulated (aspartate) and a few downregulated (creatine, choline and glycine) metabolite levels. This work revealed that observed teratogenic ailments and resulting metabolic profiles have a definite correlation, demonstrating the suitability of this procedure for testing teratogenicity, which may give a new dimension to the prioritization of lists having a large number of chemicals to be tested for their teratogenicity. Malformations observed among the full term pups also validated the findings from the embryos.