Metformin-induced endocrine disruption and oxidative stress of Oryzias latipes on two-generational condition
Introduction
Pharmaceuticals have an essential role in treating human disease and improving the production capacity in livestock [1,2]. Therefore, these pharmaceuticals have been entering the environment for decades, generally through wastewater treatment plants (WWTPs), and sometimes directly without appropriate treatment [2,3]. Consequently, in both the aqueous environment and effluent/influent of WWTP, over 600 pharmaceuticals are found at ng/L to μg/L levels [4].
Despite the societal benefits of pharmaceuticals, some studies have reported that pharmaceuticals adversely affect aquatic organisms, causing reproductive toxicity through endocrine disruption, resistance to antibiotics of fish and others, etc. [5,6]. Metformin has been prescribed globally since 1958, so it has been observed ubiquitously in ambient water and WWTP water [[7], [8], [9], [10], [11]]. The maximum concentration for WWTP influent was 129 μg/L in Germany [12]. 47 μg/L was monitored as the highest metformin concentration in WWTP effluent in Wisconsin, USA [9]. In surface water, the median value of 0.11 μg/L metformin (maximum: 0.15 μg/L) was found in the USA streams during 1999 and 2000 [13] and the median value of 0.072 μg/L (maximum: 0.19 μg/L) was monitored in the surface water of Malaysia [14]. The highest metformin concentration of surface water was 20 μg/L (median: 0.61 μg/L) in Tianjin, China [11,15], showing that in the surface water, metformin was found from non-detection level to about 20 μg/L.
AMP-activated protein kinase (AMPK) is activated by metformin via suppressing of the respiratory chain of mitochondria complex I (MRC I), lowering glucose levels in the serum by inhibiting hepatic gluconeogenesis in the liver [[16], [17], [18], [19]]. Metformin-induced AMPK activation can affect sterol biosynthesis and the hypothalamus-pituitary-gonadal (HPG) axis, which is important to the reproductive system [20]; it is presumed that metformin affects reproduction. Actually, in a previous report, metformin exposure evoked vitellogenin (VTG) transcript increase in male fathead minnow (Pimephales promelas) [21]. The significant expression of genes such as ER α and VTG in juvenile P. promelas was found, in response to metformin exposure [22]. Also, 320-day metformin treatment caused intersex gonad development, male fish size decrease and fecundity decrease in P. promelas [23], suggesting that metformin could cause an adverse effect upon a reproductive system of fish. But, there has been no report for multi-generational toxicity to a reproductive system and female fish gonad. Also, these studies were conducted only for P. promelas.
In addition to these reproductive toxicities, metformin causes oxidative stress. For instance, metformin caused oxidative stress in adipocyte [24] and in MCF-7 cell, metformin lead to ROS expression and cell viability decrease [16]. However, because MRC I is one of the sources of ROS production, the effects of metformin can be complicated with the decrease of ROS production. For example, metformin increases AMPK activity, ending in reduction of ROS yield of podocytes [19,25]. But, so far, for an aquatic ecosystem, oxidative stress occurrence by metformin has not been reported, especially on a multi-generational condition.
At 30℃, for 208-hour, about 10% of metformin hydrochloride was decomposed in water, showing that metformin would be stable for at least 9 days in water [26]. Also, 1.3∼8.2% of metformin hydrochloride dissipation suggest that metformin biodegradability might be slow in the aquatic environment [27]. These findings indicate that non-biodegradability of metformin in the aquatic environment can be chronic pressure to aquatic organisms. However, metformin has a low log Kow (−2.64) and in the repeated oral treatment test to human, it did not cause accumulation [27]. Thus, through food-chain, bioconcentration potential of it is low in the aquatic environment.
Furthermore, to precisely understand the toxicity of a chemical in an aquatic ecosystem, all possible conditions of exposure should be considered. One such condition is multi-generational exposure condition. Previously, chemical toxicity can change significantly in aquatic organisms from one generation to the next. The toxicity of a perfluoroalkyl acid mixture to Oryzias latipes was greater for offspring than for parents [28]. The perfluorooctane sulfonate (PFOS) toxicity to Daphnia magna was larger in offspring than parents [29]. However, mentioned before, there have been no chronic and multi-generational metformin exposure studies.
Together, as mentioned, metformin is distributed ubiquitously in the aquatic environment, and it can cause oxidative stress and reproductive toxicity through endocrine disruption in organisms, suggesting that metformin toxicity can be a world-wide threat. Therefore, based on diverse exposure scenarios such as the multi-generations and flow-through exposure condition, an aquatic toxicity study is required. To evaluate metformin-induced toxicity under a multi-generational exposure regimen, we monitored reproductive toxicity and oxidative stress markers in Oryzias latipes, which is a well-known test species. As reproductive toxicity markers, fecundity, hatchability, histological change of gonad, the gene expression levels of VTG1/2, ERα/β1/β2, and CYP19a were analyzed. To analyze oxidative stress, we measured analyzed biomarkers such as GSH content. Additionally, we analyzed histological changes in the brain, liver, kidney, gills, and thyroid. This study sought to inform not only the metformin regulation in the aqueous environment, but also to deepen our understanding of toxicity mechanisms caused by metformin.
Section snippets
Chemicals
Metformin (1,1-dimethylbiguanide hydrochloride) and test chemicals applied to this work were obtained from the commercial products (Sigma-Aldrich Co., MO, USA).
Fish maintenance
Test species were provided from NIER aquarium. The fish maintenance was carried out as following condition: 7.5 ± 0.2 of pH, 7–8 mg/L of dissolved oxygen (DO), 24 ± 1 ℃ of water temperature, 16h-ligh/8h-dark of photoperiod, and 55∼57 mg/L (as CaCO3) of hardness. Tetramin powder (Tetra Co., Germany) and brine shrimp at about 1% of body
Fecundity, hatchability, and metformin concentration maintenance
During 19-week metformin exposure, the measured concentrations for the 40, 120, and 360 μg/L metformin groups were 49.86 ± 2.46, 139.96 ± 6.6, and 373.06 ± 16.8 μg/L, respectively. The deviation of the measured middle and high concentrations from their nominal concentration was below 20%. Also during the exposure, lethality was not found to exceed 10 percentage in all test groups, indicating that the test conditions were appropriately sub-lethal. In response to metformin, O. latipes did not
Discussion
The treatment with endocrine disrupting chemicals (EDCs) caused significant changes in VTG, ERs, CYP19a gene expression levels and intersex occurrence [28,[38], [39], [40]]. Oryzias latipes treated in the laboratory with the known EDC, 17α-ethynylestradiol (EE2) exhibited VTG expression and intersex occurrence [28,41]. Metformin could activate AMPK by inhibiting MRC I and thereby affects HPG axis, resulting in reproductive system disturbance [20,42]. In rat pituitary cells, metformin reduced
Conclusions
Under a two-generational exposure condition, metformin caused an endocrine disruption in F0 male and female O. latipes, with intersex in F0 and F1 female fish. Metformin treatment triggered oxidative stress in F0 male fish but there was no evidence for transferring of the effect to F1 generation. In F0 female fish, only CAT activity was increased, suggesting that sexual difference may exist in oxidative stress occurrence. Together, this study demonstrated that metformin has adverse effects
Declaration of interest
All authors have no potential conflict for the publication of this study result.
Acknowledgements
This work was supported by a grant from the National Institute of Environmental Research (NIER), Funded by the Ministry of Environment (MOE) of the Republic of Korea (ex : NIER-2017-01-01-009)
References (62)
- et al.
Pharmaceuticals and personal care products (PPCPs) in the freshwater aquatic environment
Emerg. Contam.
(2017) - et al.
Pharmaceuticals and personal care products found in the Great Lakes above concentrations of environmental concern
Chemosphere
(2013) - et al.
Occurrence of the antidiabetic drug metformin and its ultimate transformation product Guanylurea in several compartments of aquatic cycle
Environ. Int.
(2014) - et al.
A global perspective on the use, occurrence, fate and effects of antidiabetic drug metformin in natural and engineered ecosystems
Environ. Pollut.
(2016) - et al.
Monitoring of 1300 organic micro-pollutants in surface waters from Tianjin, North China
Chemosphere
(2015) - et al.
Reducing insulin resistance with metformin: the evidence today
Diabetes Metab.
(2003) - et al.
Age-dependent effects in fathead minnows from the anti-diabetic drug metformin
Gen. Comp. Endocrinol.
(2016) - et al.
Emerging wastewaer contaminant metformin causes intersex and reduced fecundity in fish
Chemosphere
(2015) - et al.
Metformin induces suppression of NAD(P)H oxidase activity in podocytes
Biochem. Biophys. Res. Commun.
(2010) - et al.
Multi-generational xenoestrogenic effects of perfluoroalkyl acids (PFAAs) mixture on Oryzias latipes using a flow-through exposure system
Chemosphere
(2017)