Profiling lipid metabolites yields unique information on sex- and time-dependent responses of fathead minnows (Pimephales promelas) exposed to 17α-ethynylestradiol

Abstract

Alterations in hepatic lipid profiles of fathead minnows (FHM) exposed to the synthetic estrogen 17a-ethynylestradiol (EE2) were determined using 1 H-NMR spectroscopy-based metabolite profiling. The exposures were conducted using either 10 ng/l or 100 ng/l EE2 via a continuous flow water delivery system. Livers were collected at 1, 4, and 8 days of the exposure and 8 days after the chemical was removed from the water (i.e. an 8 day depuration). The exposure resulted in a number of sex-specific changes in lipid profiles that were also highly time dependent. Those metabolites most affected by exposure included phosphatidylcholine, diglycerides, triglycerides and cholesterol. In addition, changes in the length and degree of unsaturation of hepatic fatty acids were observed. Lipid profiles in plasma for fish collected on the 4th day of exposure were also analyzed in order to provide further insights into changes observed in hepatic metabolite changes. Using validated partial least-squares discriminant analysis (PLS-DA), the response trajectories of the male liver lipid profiles at both exposure concentrations were compared. This analysis indicated that the males exposed to the low concentration of EE2 (10 ng/l) were largely able to recover from the exposure once the chemical was removed from the water. Conversely, the males exposed to the high concentration (100 ng/l) did not appear to recover from the exposure despite the 8 day depuration.

1H NMR-based metabolomic observation of a two-phased toxic mode of action in Eisenia fetida after sub-lethal phenanthrene exposure

“…3) highlight these metabolites and the extent that the metabolic response varies with exposure to increasing phenanthrene concentration. [10,38] The percentage change of the identified metabolites relative to the control (Fig. 4) suggests that metabolic changes are also related to exposure concentration over the range of concentrations studied.…”

“…The percentage changes in the intensity of metabolite peaks of exposed worms relative to the controls were obtained by first subtracting the buckets that pertain to the metabolites in the control earthworms from the exposed earthworms and then dividing again by the buckets in the control earthworms. [10,38] The percentage changes that were significantly different from the control are labelled with an asterisk (*; at a ¼ 0.05). The percentage changes are shown with their associated standard error.…”

“…A trajectory of responses to exposure of a chemical can signify its MOA, as exposure to chemicals with varying toxicities leads to trajectories that differ in their geometries or the overall shape of the curve. [10,11,38,49,50] The trajectory of the metabolic profile of E. fetida is shown in Fig. 5a.…”

“…Difference class 1 H NMR spectra were constructed to identify metabolites that had significantly increased or decreased relative to the control. [10,38] The buckets generated by AMIX 3.8.4 statistics tool, which represents the binned 1 H NMR spectra of E. fetida extracts were then imported into Microsoft Excel (version 12.0.6504, Microsoft Corporation, Redmond, WA). A Levene's test performed on the buckets revealed that there was equal variance at a ¼ 0.05.…”

“…The buckets representing metabolites peaks that were not statistically significant from the controls were then replaced with a zero resulting in a ttest filtered 1 H NMR difference spectrum. [10,38] The buckets were then imported into ACD/1D NMR manager (Advanced Chemistry Development, version 12.0, Toronto, Canada) to acquire the difference spectra. The percentage changes in the intensity of metabolite peaks of exposed worms relative to the controls were obtained by first subtracting the buckets that pertain to the metabolites in the control earthworms from the exposed earthworms and then dividing again by the buckets in the control earthworms.…”