Elsevier

Journal of Hazardous Materials

Volume 367, 5 April 2019, Pages 171-181
Journal of Hazardous Materials

Metformin-induced endocrine disruption and oxidative stress of Oryzias latipes on two-generational condition

https://doi.org/10.1016/j.jhazmat.2018.12.084Get rights and content

Highlights

  • Oryzias latipes were exposed to metformin for two generations.

  • F0 males had increased expression of CYP19a and ERα.

  • Females had decreased expression of VTG2 and ERβ1 with intersex occurrence.

  • Metformin increased reactive oxygen species abundance.

  • Metformin led to oxidative stress and two-generational endocrine disruption.

Abstract

Metformin has been treated for diabetes (type 2). Nowadays, this compound is frequently found in ambient water, influent/effluent of a wastewater treatment plant. To evaluate the metformin aquatic toxicity under a multi-generational exposure regimen, we exposed Oryzias latipes to metformin for two generations (133 d) and investigated its adverse effects. In the F0 generation, metformin significantly elevated gene expression for cytochrome P450 19a (CYP19a) and estrogen receptor α (ERα) in male fish; in female fish, the treatment decreased gene expression of vitellogenin (VTG2) and ERβ1, suggesting endocrine disruption (one-way ANOVA, p <  0.05). Intersex occurrence of F0 female fish were found in a concentration-dependent manner, whereas no significant changes in fecundity and hatching rate were observed (p <  0.05). Metformin increased the reactive oxygen species (ROS) content, and decreased the glutathione (GSH) content in F0 male fish compared with those of the control (one-way ANOVA, p >  0.05). In F0 female fish, metformin increased catalase activity compared with that of the control (p >  0.05). The results demonstrated that metformin leads to oxidative stress and two-generation endocrine disruption in O. latipes. These results may be useful for better understanding metformin toxicity mechanism.

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)

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