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 chemicaland 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.

Are Fish Populations at Risk? Metformin Disrupts Zebrafish Development and Reproductive Processes at Chronic Environmentally Relevant Concentrations

“…Although short-term reproductive studies with adult P. promelas exposed in pairs failed to show MET disruption in fish reproduction, , these studies may not represent the full effects of environmental exposure, since the critical period of sexual differentiation was not included in the exposure. Despite the short-term reproductive studies referenced above, the assumption that pairwise breeding is a better alternative to the tank breeding approach to assess MET effects in fish reproductive success is largely in agreement with the two full life cycle studies that addressed the reproductive output of P. promelas exposed to MET. , Even though Parrot et al (2021) failed to find MET adverse effects in the reproductive success of P. promelas using a method similar to our tank breeding approach, Niemuth and Klapper (2015) observed decreased fecundity in MET exposed fish with a pairwise breeding approach.…”

Is the Environmental Risk of Metformin Underestimated?

“…However, several works performed by distinct institutions using different fish species point mostly to negative effects at ranges from nanograms per liter to low micrograms per liter. Thus, even if some contrasting findings were reported by different teams for fathead minnows ( Pimephales promelas ), − with the latest study by Nielsen et al showing the impact on embryonic development at 5 μg/L, particularly in wild-spawned fathead minnows, most studies support effects in the range of environmental concentrations. The reported effects include the disruption of embryonic development in zebrafish and fathead minnows at concentrations as low as 0.39–5 μg/L; − vitellogenin (VTG) induction and disruption of reproduction-associated signaling pathways in fathead minnows, zebrafish, and Japanese rice fish ( Oryzias latipes ) − at concentrations ranging from 0.39 to 40 μg/L; and changes in the metabolism of lifelong-exposed zebrafish and in processes related to the cell cycle, DNA repair, and steroid hormone biosynthesis, concomitantly to alterations in apical end points such as growth, hepatosomatic index, and fertilization rate , at concentrations ranging from 0.39 to 14.4 μg/L.…”

Assessing Contaminants of Emerging Concern in the Great Lakes Ecosystem: A Decade of Method Development and Practical Application

“…In addition, the types of animals amenable for laboratory testing are limited. Despite these challenges, important information about the biological effects of CECs has been assembled through laboratory exposures of fathead minnows, largemouth bass ( Micropterus salmoides ), bluegill sunfish ( Lepomis macrochirus ), and plain pocketbook mussels ( Lampsilis cardium ) found in Great Lakes tributaries (Blackwell et al, 2022; Cipoletti et al, 2019; Gill et al, 2022). For example, the microbiota of the largemouth bass gut was unaltered by exposure to a mixture of agricultural CECs commonly found in Great Lakes tributaries (Gill et al, 2022).…”

“…Not only were both frequently present at elevated concentrations, but some reports in the literature suggested that metformin may cause reproductive endocrine disruption in fish (Niemuth & Klaper, 2015). To address the dearth of high‐quality effects data, comprehensive studies were undertaken to assess possible endocrine and reproductive toxicity of the two chemicals (Blackwell et al, 2022). Neither metformin nor guanylurea appeared to be endocrine‐active or to cause reproductive dysfunction, even at concentrations far exceeding those observed in the GLRI samples.…”