Metabolomics for informing adverse outcome pathways: Androgen receptor activation and the pharmaceutical spironolactone

One objective in developing adverse outcome pathways (AOPs) is to connect biological changes that are relevant to risk assessors (i.e., fecundity) to molecular and cellular-level alterations that might be detectable at earlier stages of a chemical exposure. Here, we examined biochemical responses of fathead minnows (Pimephales promelas) to inform an AOP relevant to spironolactone's activation of the androgen receptor, as well as explore other biological impacts possibly unrelated to this receptor. Liquid chromatography with high resolution mass spectrometry (LC-MS) was used to measure changes in endogenous polar metabolites in livers of male and female fish that were exposed to five water concentrations of spironolactone (0, 0.05, 0.5, 5, or 50 μg L−1) for 21 days. Metabolite profiles were affected at the two highest concentrations (5 and 50 μg L−1), but not in the lower-level exposures, which agreed with earlier reported results of reduced female fecundity and plasma vitellogenin (VTG) levels. We then applied partial least squares regression to assess whether metabolite alterations covaried with changes in fecundity, VTG gene expression and protein concentrations, and plasma 17β-estradiol and testosterone concentrations. Metabolite profiles significantly covaried with all measured endpoints in females, but only with plasma testosterone in males. Fecundity reductions occurred in parallel with changes in metabolites important in osmoregulation (e.g., betaine), membrane transport (e.g., l-carnitine), and biosynthesis of carnitine (e.g., methionine) and VTG (e.g., glutamate). Based on a network analysis program (i.e., mummichog), spironolactone also affected amino acid, tryptophan, and fatty acid metabolism. Thus, by identifying possible key events related to changes in biochemical pathways, this approach built upon an established AOP describing spironolactone's androgenic properties and highlighted broader implications potentially unrelated to androgen receptor activation, which could form a basis for the development of an AOP network.