Metabolite profiles of repeatedly sampled urine from male fathead minnows (Pimephales promelas) contain unique lipid signatures following exposure to anti-androgens

The purpose of this study was twofold. First, we sought to identify candidate markers of exposure to anti-androgens by analyzing endogenous metabolite profiles in the urine of male fathead minnows (mFHM, Pimephales promelas). Based on earlier work, we hypothesized that unidentified lipids in the urine of mFHM were selectively responsive to exposure to androgen receptor antagonists, which is otherwise difficult to confirm using established fish toxicity assays. A second goal was to evaluate the feasibility of non-lethally and repeatedly sampling urine from individual mFHMs over the time course of response to a chemical exposure. Accordingly, we exposed mFHM to the model anti-androgens vinclozolin or flutamide. Urine was collected from each fish at 48 hour intervals over the course of a 14 day exposure. Parallel experiments were conducted with mFHM exposed to bisphenol A or control water. The frequent handling/sampling regime did not cause apparent adverse effects on the fish. Endogenous metabolite profiling was conducted with gas chromatography–mass spectrometry (GC–MS), which exhibited lower variation for the urinary metabolome than was found in earlier work with nuclear magnetic resonance (NMR) spectroscopy. Specifically, for inter- and intra-individual variations, the median spectrum-wide relative standard deviation (RSD) was 32.6% and 33.3%, respectively, for GC–MS analysis of urine from unexposed mFHM. These results compared favorably with similar measurements of urine from other model species, including the Sprague Dawley rat. In addition, GC–MS allowed us to identify several lipids (e.g., certain saturated fatty acids) in mFHM urine as candidate markers of exposure to androgen receptor antagonists.