Effects of Metformin and its Metabolite Guanylurea on Fathead Minnow (Pimephales promelas) Reproduction

Abstract

Metformin, along with its biotransformation product guanylurea, is commonly observed in municipal wastewaters and subsequent surface waters. Previous studies in fish have identified metformin as a potential endocrine-active compound, but there are inconsistencies with regard to its effects. To further investigate the potential reproductive toxicity of metformin and guanylurea to fish, a series of experiments was performed with adult fathead minnows (Pimephales promelas). First, explants of fathead minnow ovary tissue were exposed to 0.001-100 µM metformin or guanylurea to investigate whether the compounds could directly perturb steroidogenesis. Second, spawning pairs of fathead minnows were exposed to metformin (0.41, 4.1, and 41 µg/L) or guanylurea (1.0, 10, and 100 µg/L) for 23 days to assess impacts on reproduction. Lastly, male fathead minnows were exposed to 41 µg/L metformin, 100 µg/L guanylurea, or a mixture of both compounds, with samples collected over a 96-h time course to investigate potential impacts to the hepatic transcriptome or metabolome. Neither metformin nor guanylurea affected steroid production by ovary tissue exposed ex vivo. In the 23 days of exposure, neither compound significantly impacted transcription of endocrine-related genes in male liver or gonad, circulating steroid concentrations in either sex, or fecundity of spawning pairs. In the 96-h time course, 100 µg guanylurea/L elicited more differentially expressed genes than 41 µg metformin/L and showed the greatest impacts at 96 h. Hepatic transcriptome and metabolome changes were chemical- and time-dependent, with the largest impact on the metabolome observed at 23 days of exposure to 100 µg guanylurea/L. Overall, metformin and guanylurea did not elicit effects consistent with reproductive toxicity in adult fathead minnows at environmentally relevant concentrations. Environ Toxicol Chem 2022;41:2708-2720. © 2022 SETAC. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA.

Keywords: Aquatic toxicology; contaminants of emerging concern; endocrine-disrupting compounds; reproductive toxicity.

FIGURE 1:

Production of 17β-estradiol (E2) or testosterone (T) from female fathead minnow ovary tissue exposed to varying concentrations of metformin, guanylurea, or prochloraz (PCZ; positive control; 5 μM) ex vivo. Values represent mean ± SEM (n = 10 individual ovary subsamples/treatment). Asterisks denote significant difference relative to controls (p < 0.05).

FIGURE 2:

Cumulative egg production/female over time (A) and number of spawns/female (B) in spawning fathead minnow pairs exposed to control Lake Superior water, metformin (MET; 0.41, 4.1, 41 μg/L), or guanylurea (GU; 1.0, 10, 100 μg/L) for 23 days. Values represent mean ± SEM (n = 9–12 pairs of fish/treatment). No significant differences were observed relative to Lake Superior water controls.

FIGURE 3:

Differentially expressed genes in liver tissue (A) and total intensity difference values of metabolite changes in polar liver extracts (B) from male fathead minnow exposed to metformin (MET), guanylurea (GU), or a mixture of both for up to 23 days. Total intensity difference values are the sum of the absolute values of peaks in t-test filtered difference spectra generated by subtracting the average nuclear magnetic resonance (NMR) spectrum of the controls from the average NMR spectrum of each exposure. Values for two other reproductive toxicants, bisphenol A (BPA) and ethinylestradiol (EE2), which were previously assessed in fathead minnow (Ekman et al., 2008a; Ekman et al., 2012), are shown for comparison.