Environmental concentrations of the antidiabetic Metformin cause liver damage in Astyanax lacustris (Lütken, 1875) individuals after chronic exposure

Environmental concentrations of the antidiabetic Metformin cause liver damage in Astyanax lacustris (Lütken, 1875) individuals after chronic exposure | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Environmental concentrations of the antidiabetic Metformin cause liver damage in Astyanax lacustris (Lütken, 1875) individuals after chronic exposure Brennda Ribeiro Paupitz, Pablo Américo Barbieri, Luara Lupepsa, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4031547/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Drugs are emerging contaminants that provide concern when it comes to the adverse effects they can cause on organisms that are not the target of therapeutic action. Because water treatment methods do not entirely remove them, they are found in worrying concentrations in the aquatic environment. Antidiabetic Metformin has been found in the environment worldwide, and studies show it has a potential endocrine disrupting effect. However, more research is needed regarding its impact on bioindicator organisms, such as fish. This work aimed to evaluate the effect of chronic exposure to Metformin in the liver, the organ responsible for the metabolism of xenobiotics, from Astyanax lacustris. The results obtained from histological sections of the organ show that Metformin induced liver damage since the number, size, and composition of hepatocytes have changed. This study demonstrates the need for more research on the damage metformin can cause aquatic life. Metformin liver hepatocyte endocrine disruptor aquatic pollution Figures Figure 1 Figure 2 1. Introduction Wastewater treatment plants have been the focus of studies regarding introducing pollutants, such as pharmaceutically active compounds, into the environment (Ghirardini et al. 2021 ). Even in non-therapeutic doses, these compounds can still interfere with the environment and human health (Daughton and Ruhoy, 2008 ). In general, these studies range from evaluating the concentration of medications in the environment to removal alternatives, which often involve the use of bioreactors (Ghirardini et al., 2021 ; Goswami et al., 2018 ; Scheurer et al., 2012 ) aiming to minimize the damages caused by these pollutants (Vatovec; Wagoner; Evans, 2017 ). Pharmaceuticals are released into the environment through two main routes. Improper disposal of medications that will no longer be used is quite common, whether due to leftover medications, their expiration, or patient non-adherence to treatment, which usually leads to the disposal of the drugs in the toilet or household trash. Another route is the excretion of the unmetabolized component through the patient's feces and urine or the release of topically used medication into bathwater (Daughton and Ruhoy, 2008 ). This disposal contributes to the pollution of groundwater and surface waters through septic systems and reaches rivers and lakes, the primary recipients (Carrara et al. 2008 ). Pharmaceuticals of high relevance are often found in low concentrations in the environment, and numerous knowledge gaps persist, underscoring the imperative for further research to comprehensively evaluate the potential risks posed by pharmaceuticals in aquatic ecosystems (Fent; Weston; Caminada, 2006 ). Among these pharmaceuticals, the antidiabetic Metformin (1,1-dimethylbiguanide), which is a first-line treatment for patients with type 2 diabetes mellitus (Scheurer; Sacher; Brauch, 2009 ; Idf, 2017), has gained significant attention. Because it is widely used worldwide and is not metabolized by the human body, it is found in its non-biotransformed form in aquatic ecosystems across diverse regions, as shown by studies (Kane, 2020; Graham et al., 2011 ). Trautwein and Kümmerer ( 2011 ) and Scheurer et al. ( 2012 ) demonstrated through biodegradation tests with microorganisms that Metformin is a drug not readily degraded in the aquatic environment. Besides, conventional wastewater treatment is inefficient in eliminating this compound (Carbuloni et al., 2020 ) In the natural aquatic environment, fish interact with biological systems and substances such as pharmaceuticals, which trigger physiological changes. Environmental pollution alters the chemical composition of natural habitats, affecting organisms that are not the target of these compounds' action (Monteiro et al., 2006 ). Adverse effects of the antidiabetic have been observed in some fish species, such as Pimephales promelas , Oryzias latipes , Betta splendens , and Astyanax lacustris . The observed changes include variations in vitellogenin levels, estrogen receptor levels, gonadotropin-releasing hormone levels, alterations in the expression of the CYP19A gene, presentation of oxidative stress, endocrine disruption, effects on animal behavior and morphological modifications in gills (Crago et al., 2016 ; Lee et al., 2019 ; Maclaren; Wisniewski; Maclaren, 2018 ; Barbieri, 2022). Among the most notable effects of Metformin is the occurrence of intersex individuals in male P. promelas , induced by activation of vitellogenin mRNA transcription, a protein responsible for vitellogenesis that usually is only expressed in females (Niemuth et al., 2015 ). The impact of xenobiotics, such as Metformin, on these organisms can be assessed by analyzing biochemical disturbances caused, allowing fish to be used as bioindicators in polluted environments (Monteiro et al., 2006 ). The liver plays an essential role in the metabolism of xenobiotics, making it susceptible to damage from these agents as hepatic tissue is vulnerable to environmental changes, often serving as a biomarker for assessing damage (Monteiro et al., 2006 ). Astyanax lacustris is considered an excellent species for experimental modeling due to its sensitivity and response to the presence of contaminants in the environment, often used as a bioindicator species (Sposito et al., 2019 ). It is widely distributed in Brazilian watersheds and holds significant ecological importance (Lucena, Soares, 2016 ; Vilella, Becker, Hartz, 2002). Based on this, the present study aimed to evaluate the potential ecotoxicological impacts of Metformin on the liver of A. lacustris fish specimens, a species with wide distribution in the neotropical region and considerable ecological significance due to its interactions with other ecosystem organisms. 2. Material and methods 2.1. Fish Acquisition and Maintenance A. lacustris individuals were obtained from commercial establishments from fish farms where the animals did not come into contact with effluents. Transportation was carried out using plastic bags containing 1/3 water and 2/3 oxygen, placed in a styrofoam box to protect against shocks and reduce fish stress. The specimens were kept in the fish vivarium located in block T-10, where they were maintained in water tanks with 150 liters containing 100 liters of water at ambient temperature and lighting conditions. They were fed with commercial feed for small fish once a day until the experimental procedures. Throughout the experiments, parameters such as water temperature, oxygen content, conductivity, and pH were consistently monitored and maintained within the optimal range for these fish, ensuring that these factors did not influence the results. The main parameters and variables required for the well-being of the animals, the stocking density, and the water exchange in the aquariums were carried out according to CONCEA Normative Resolution No. 34, dated July 27, 2017. 2.2. Preparation of Metformin Stock Solution For the experiments, two sources of Metformin were used: metformin sigma P.A (used for animals exposed to concentrations in the microgram range) and commercial Medley metformin (used for animals exposed to milligram concentrations). A stock solution with a concentration of 7800 mg/L was prepared from commercial Medley metformin (metformin hydrochloride 780 mg + excipients q.s.p). The tablets were crushed using a mortar and pestle and diluted in distilled water. A stock solution with a concentration of 50 µg/L was prepared from metformin sigma P.A. Both solutions were aliquoted and stored in a refrigerated and dark location. 2.3. Metformin Exposure Juvenile male animals (30 to 40 days post-hatch) were randomly selected and exposed for 90 days. The group included a control subgroup and four additional subgroups based on the concentration of Metformin: 50 µg/L, 100 µg/L, 1000 µg/L, and 10,000 µg/L (Table 1 ). The distribution of fishes among the concentration groups was carried out following the blinding methods. The concentrations of 50 µg/L and 100 µg/L were established based on concentrations found in surface waters of different aquatic environments, using values close to the minimum and maximum (Scheurer et al., 2009 ; Scheurer et al., 2012 ). The concentration of 10,000 µg/L corresponds to the NOEC value for zebrafish in chronic tests (Moermond and Smit, 2015 ). The 1 mg/L concentration was used to test a concentration ten times lower than the NOEC. Table 1 Group 1: Juvenile fish aged 30 to 40 days post-hatching. Chronic treatment (90 days): 5 animals for each concentration + control. Subgroups Group 1 2 3 4 5 Total Concentration 50 ug/L 100 ug/L 1000 µg/L 10.000 µg/L Controle Animals 5 5 5 5 5 25 2.4. Euthanasia Method for Animals Each animal was immersed in a clove oil/eugenol solution after being removed from the aquarium, following the instructions of Inoue and Morais (2007). The anesthetic solution was prepared: 1 ml of the anesthetic (eugenol) diluted in 3 ml of alcohol. Subsequently, 0.5 ml of this solution was added to each 1 liter of water. The fish were handled only after no reaction to any physical stimulus, indicating death by anesthetic overdose. The ethics committee (CEUA/UEM) approved the animal experimentation protocol under number 6409140218. 2.5. Histological Analysis Liver samples were washed in 0.9% saline solution and fixed in aqueous Bouin's solution for 12 hours (Behmer et al., 1976 ), then stored in 70% alcohol. The material was dehydrated in increasing series of alcohols (80%, 90%, and 100%), cleared in xylene, and embedded in paraffin for histological processing. Semi-serial transverse sections of 5µm thickness were obtained using a LEICA rotary microtome at the Animal Histotechnique Laboratory of the Department of Morphological Sciences at Universidade Estadual de Maringá. Slides were stained using the Hematoxylin-Eosin (H.E.) method (Behmer et al., 1976 ). The light microscope (Olympus CX31RBSFA) was used to verify morphological analysis and liver alterations. All analyses were conducted using the experimental blinding method, where it was not possible to know which group the analyzed fish belonged to. Additionally, the samples were analyzed by two different researchers to ensure the same result. 2.6. Quantitative Analysis of Hepatic Cell Numbers and Morphometric Analysis of Hepatocyte Cytoplasm and Nucleus Images of H.E.-stained sections were captured using an Olympus BX41® optical microscope (40x objective) with an Olympus Q-Color® 3 camera, connected to a computer with Q-Capture® software. In the hepatocyte counting analysis, 50 images/animal were measured, totaling 250 images/group. For morphometry, 200 hepatocytes/animal were measured, totaling 1000 hepatocytes/group. The Image Pro-Plus® 4.5 software (Media Cybernetics) was used for morphometric analyses. The obtained data were checked for normality using the Kolmogorov-Smirnov test. Subsequently, they were subjected to a one-way analysis of variance (ANOVA) followed by Tukey's post-test. The significance level adopted was 5%, and the results were expressed as mean ± standard error. The statistical program used was GraphPad Prism version 8. 3. Results Morphologically, the livers of the control animals of A. lacustris displayed polygonal hepatocytes with cytoplasmic vacuolation, centrally located nuclei with evident nucleoli (Fig. 1a) similar to what has been described for other characids (Petcoff et al., 2006 ; Marcon et al., 2015 ). The vacuoles appeared typically flocculent, angular with smooth edges, and minimal displacement of the nucleus, characteristic of vacuoles that store glycogen. In control animals, an eosinophilic staining of the hepatocellular cytoplasm was observed (Fig. 2A), whereas in animals exposed to Metformin, increased basophilia was noted in the hepatocyte cytoplasm (Figs. 2B, 2C, 2D). In animals exposed to the drug, it was also possible to observe hydropic hepatocytes, characterized by diffuse cytoplasmic clearing, maintenance of the nucleus in a central position, and architectural distortion of hepatocyte cords with compression of the sinusoids. Another aspect noticed was increased eosinophilic fluid in several blood vessels (Figs. 2B, 2C, 2D - arrowhead). Figure 1 - Photomicrograph of A. lacustris liver chronically exposed to Metformin. (A) Control; (B) 50 µg/L concentration; (C) 100 µg/L concentration; (D) 1000 µg/L concentration; (E) 10,000 µg/L concentration. Arrows indicate cytoplasmic vacuolization in hepatocytes. Arrowheads indicate the presence of proteinaceous fluid within blood vessels. Only in treated animals is a diffuse aspect of hydropic hepatocytes observed. There was a decrease in the number of these cells compared to the control-exposed individuals (Fig. 1b and 2a). The animals in the control group had an average of 150 hepatocytes per field/image, while at the concentration of 50 µg/L, this value dropped to an average of 125 hepatocytes per field/image. At concentrations of 100 µg/L, 1000 µg/L, and 10,000 µg/L, the cell count was even lower, with an average of 80 hepatocytes per field/image. Statistically, the data from all concentrations showed a significant effect (p > 0.05) compared to the control. Based on the cell count result, morphometry of the cytoplasmic area of the cells was performed to corroborate the previous analysis. Compared to the control, there was a progressive increase in cytoplasm size according to the metformin concentration (Fig. 1B), demonstrating a dose-dependent relationship. Statistically, the results showed a significant effect (p 0.05) in nucleus size according to the dose increase, as observed with the cytoplasm (Fig. 1C). The concentration that exhibited the largest nuclear area was 10,000 µg/L, corresponding to the NOEC concentration. Figure 2 - Graph depicting a comparison of treated groups with the control group. (a) Hepatocyte count, (b) Cytoplasmic morphometric analysis, and (c) Nuclear morphometric analysis. * - Significant difference compared to control (p < 0.05). Vacuolization was found in the cytoplasm of hepatocytes in the control animals, and it was more pronounced in the hepatocytes of fish exposed to Metformin (Fig. 2). 4. Discussion This study conducted a quantitative and qualitative analysis of the structural parameters of hepatocytes in the liver of an experimental model exposed to Metformin, A. lacustris, revealing alterations such as decreased cell count, progressive cytoplasmic and nuclear enlargement, and demonstrating dose dependence. Furthermore, other significant changes occurred in liver cells, including vacuole size, increased basophilia in the cytoplasm, architectural distortion of hepatocyte cords with sinusoidal compression, and increased eosinophilic fluid content in various blood vessels. These findings indicate profound histochemical alterations in the hepatocytes of these animals. While the physiological and structural liver structure is highly conserved among vertebrates, the liver of teleost fish is susceptible to environmental changes, whether in natural ecosystems or laboratory exposures, exhibiting both morphological and physiological changes in response to contamination (Wolf and Wheeler, 2018 ; Santos et al., 2013 ). Histopathological analysis is often required to assess and determine liver damage presence or absence under toxicological evaluation (Wolf and Wolfe, 2005 ). Our results demonstrate that Metformin, a significant emerging contaminant in wastewater and surface waters, significantly impacts the liver of A. lacustris at environmentally relevant concentrations. Metformin, based on the PEC/PNEC value, has been considered a low environmental risk drug (Caldwell et al., 2019 ), where PEC is the predicted environmental concentration for a specific environment, and PNEC is the predicted no-effect concentration, below which adverse effects are unlikely to occur during exposure. The PNEC for chronic toxicity is typically estimated using the NOEC - no observed effect concentration (Johnson et al., 2009). The established NOEC value for Metformin is 10 mg/L for Danio rerio , and PNEC is 1 mg/L (Caldwell et al., 2019 ), meaning that this value corresponds to the highest concentration of the substance that does not cause statistically significant adverse effects in organisms under the test exposure time and conditions. However, even at concentrations below the described PNEC value for Metformin, the results of this study demonstrate the occurrence of histopathological alterations in the liver. Moreover, a dose-dependent trend was observed in the reported morphometric effects, as higher concentrations of the compound in the water led to more significant results. Based on our data, we can conclude that a chemical substance does not cause death in aquatic organisms in toxicity tests does not indicate that it does not cause pathological damage to them. The alteration from eosinophilic staining of the hepatocellular cytoplasm in control animals to increased basophilia in treated animals observed in this study may be correlated with vitellogenin production, a protein responsible for oocyte development in females. According to the OECD document guiding diagnosing histopathological changes in fish exposed to potential endocrine disruptors (Johnson et al., 2009), a diffuse increase in hepatocellular basophilia is observed in animals exposed to a compound with estrogenic activity. This increase in basophilia, associated with increased vitellogenin production, presumably mimics the expanded metabolic state (e.g., increased endoplasmic reticulum) required for vitellogenin production in reproductively active females. Van den Belt-K et al. (2002) also detected hepatocellular basophilia in female and male zebrafish exposed to an estrogen analog (ethinyl estradiol), indicating a high mRNA content. This hepatocyte basophilia coincides with high plasma vitellogenin levels, showing active vitellogenin synthesis in the liver. According to the authors, in exposed males but not in control animals, capillaries (and other vessels) are dilated and filled with eosinophilic plasma, indicative of high protein content (vitellogenin). Similarly, our results also showed an increase in the amount of eosinophilic protein fluid in various blood vessels. The increase in intracellular fluid, characterizing hydropic hepatocytes, also known as hydropic degeneration or cellular edema, is the first manifestation of nearly all forms of cellular damage. It is a reversible, non-lethal alteration. Hydropic degeneration is the accumulation of water within the intracellular environment, resulting from imbalances in osmotic gradient control at the level of the cytoplasmic membrane and mechanisms of absorption, elimination of water, and intracellular electrolytes. Intracellular water accumulation occurs due to the cell's inability to maintain ionic balance and fluid homeostasis, resulting from failures in energy-dependent pumps of cell membranes (Miller and Zachary, 2017 ). The correlation between Metformin and decreased ATP production, leading to the losses, occurs due to the inhibition of Complex I of the mitochondrial respiratory chain (Faure et al., 2018 ). Consequently, the decline in intracellular ATP could interfere with ion transport through the Na+/K+-ATPase and thus disrupt the osmotic balance of hepatocytes in animals exposed to Metformin, explaining the observation of hydropic hepatocytes. Analyzing A. lacustris exposed to Metformin, previously conducted by our team, also revealed cellular changes associated with decreased intracellular ATP, such as the presence of echinocytes seen in blood smears under light microscopy and loss of microdigitations observed under scanning electron microscopy in gills, both caused by cytoskeleton modulations involving dissociations between spectrin-actin, which require energy for stability. The morphometric parameters of the cytoplasm and nucleus showed increased volume with increasing metformin dose. This growth reflects a response found by Macêdo et al. ( 2020 ) in A. lacustris exposed to river waters from different regions containing various contaminants, mainly metals, demonstrating that exposure of this species to environmental pollutants results in this hepatic response. According to Wolf and Wolfe ( 2005 ), the increase in cytoplasmic volume found in hepatocytes can be caused by hypertrophy due to organelle proliferation (hypertrophy), failure in the mitosis process causing megalocytosis, and osmotic imbalance leading to increased intracellular water (hydropic degeneration or edema) and cytoplasmic vacuolization. Based on the characteristics observed in A. lacustris hepatocytes, it is assumed that the increased cell volume occurred due to hydropic degeneration and glycogen accumulation in the vacuoles. Hepatic glycogen content increased in Salmo trutta f. fario exposed to environmentally relevant concentrations of Metformin (Jacob et al., 2018 ). Based on the increase in cytoplasmic and nuclear volume with increasing antidiabetic concentration, it is presumed that the observed decrease in cell count per field/image occurred due to increased cell volume rather than a decrease in hepatocyte number. However, the hepatosomatic index should be analyzed to confirm this hypothesis. In addition to the increase in cytoplasmic volume, there was an increase in nuclear size compared to the control subgroup. In the work of Santos et al. ( 2013 ), brown trout individuals, also teleosts, exposed to a hepatocarcinogenic initiator, N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), exhibited hepatocytes with a larger nuclear volume than "normal" hepatocytes, indicating that the nucleus of the healthy hepatocyte differs structurally and functionally from that of the exposed hepatocyte, similar to our results. The increase in nuclear volume observed here may be associated with increased expression of genes related to protein synthesis, as studies have already shown overexpression of genes used as markers of damage caused by endocrine disruptors. The egg yolk protein, vitellogenin (VTG), consists of a polypeptide whose increased levels in male fish have been attributed to hepatotoxic responses mediated by contaminants lacking hormonal structure but with potential endocrine disruptor effects, serving as an essential biomarker (Baumann et al., 2020 ). Niemuth et al. ( 2015 ) found in their study with P. promelas exposed to environmentally relevant concentrations of Metformin for 28 days that there was overexpression of VTG mRNA in males of the species, while levels in exposed females did not differ from the control. Although insignificant, Plasma VTG levels in males were higher than in the control. The increase in VTG mRNA expression was also observed in male P. promelas exposed to another contaminant, the synthetic estrogen 17α-ethinylestradiol (EE2) (Lattier et al., 2002 ). 5. Conclusion We observed that A. lacustris individuals exposed to different concentrations of metformin, including environmentally relevant concentrations, exhibited significant alterations in liver tissue compared to the control group. This research demonstrates the environmental relevance of metformin as a contaminant. Due to its widespread use worldwide and persistence in the aquatic ecosystem, this compound needs to be studied as a potential emerging environmental contaminant, considering the obtained results here. Declarations CONFLICT OF INTEREST Authors declare that they do not have any conflict of interest. Ethics approval Ethical approval for this study was obtained from Ethics Committee on the Use of Animals (CEUA - State University of Maringá, Paraná, Brazil) under decision number 6409140218. FUNDING INFORMATION This study was financed by CAPES (Coordenadoria de Aperfeiçoamento de Ensino Superior). Author Contribution All authors discussed the results and contributed to the final manuscript. Luciana Andreia Borin-Carvalho, Brennda Ribeiro Paupitz and Ana Luiza de Brito Portela-Castro conceived of the presented idea. Material preparation, data collection and analysis were performed by Brennda Ribeiro Paupitz, Luara Lupepsa and Pablo Americo Barbieri. The manuscript was written by Brennda Ribeiro Paupitz with support from Luciana Andreia Borin-Carvalho, Ana Luiza de Brito Portela-Castro. All authors read and approved the final manuscript. ACKNOWLEDGEMENTS We thank of the Centro de Microscopia (CMI) of Complexo de Centrais de Apoio à pesquisa (COMCAP) at the Universidade Estadual de Maringá (UEM), Maringá, PR in processing material used and assistance in handling the equipment. This work was supported by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES). 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Occurrence and fate of the antidiabetic drug metformin and its metabolite guanylurea in the environment and during drinking water treatment. Water Research, 46, 4790-4802. Doi: 10.1016/j.watres.2012.06.019. Scheurer, M., Sacher, F, Brauch, HJ, 2009. Occurrence of the antidiabetic drug metformin in sewage and surface waters in Germany. Journal of Environmental Monitoring, 11, 9. doi:10.1039/b909311g Sposito, J.C.V., Francisco, L F V, Crispim, BA, Dantas, FGS, Souza, JP, Viana, LF, Solórzano, JCJ, Oliveira, KMP, Barufatti, A, 2019. Influence of Land Use and Cover on Toxicogenetic Potential of Surface Water from Central-West Brazilian Rivers. Archives of Environmental Contamination and Toxicology, 76, 483-495. Trautwein, C., Kümmerer, K, 2011. Incomplete aerobic degradation of the antidiabetic drug Metformin and identification of the bacterial dead-end transformation product Guanylurea. Chemosphere, 85, 5, 765-773. Doi: 10.1016/j.chemosphere.2011.06.057. Van Den Belt, K., Wester, PW, Van Der Vem, LTM, Verheyen, R, Witters, H. Effects of ethynylestradiol on the reproductive physiology in zebrafish ( Danio rerio ). Environmental Toxicology and Chemistry, n. 21, v. 4, p. 767-775, 2002. Vatovec, C., Wagoner, EV, Evans, C, 2017. Investigating sources of pharmaceutical pollution: Survey of over-the-counter and prescription medication purchasing, use, and disposal practices among university students. Journal of Environmental Management, 198, 348–352. doi:10.1016/j.jenvman.2017.04.101. Vilella, F.S., Becker, FG, Harts, SM, 2002. Diet of Astyanax species (Teleostei, Characidae) in an Atlantic Forest River in Southern Brazil. Brazilian Archives of Biology and Technology, 45, 2, 223-232. https://doi.org/10.1590/S1516-89132002000200015 Wolf, J.C., Wolfe, MJ, 2005. A brief overview of nonneoplastic hepatic toxicity in fish. Toxicologic Pathology, 33, 1, 75-85. doi: 10.1080/01926230590890187. Wolf, J.C., Wheeler, JR, 2018. A critical review of histopathological findings associated with endocrine and non-endocrine hepatic toxicity in fish models. Aquatic Toxicology, 197, 60-78. Doi: 10.1016/j.aquatox.2018.01.013 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4031547","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":277284284,"identity":"644d2de9-0a47-49fe-b750-25f4c670045e","order_by":0,"name":"Brennda Ribeiro Paupitz","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA7UlEQVRIiWNgGAWjYBACAzDJxiBnf4D5GITNTqQWY4YDbGkMDAlANjORWhIbDvCYgbUwENJiLn342OOCMrvExgaebw8+/tgmz8fMwPjhYw5uLZZ9aenGM84lGzcz8G43nJFw27CNmYFZcuY2PA47w2MmzdvGLNvGwLtNmifhNiNQCxszL14t/N+AWuoZexh4noG02BOhhYcNqOWw4gwGIAOoJZGgFsseNjNpnnPHjQ0Y2MwkZ6TdTm5jZmzG6xdzHmage8qq5QwYmJ9JfLC5bTu/vfngh494tCCA/AMYi7GBGPWjYBSMglEwCvAAAOqyRKodNzD0AAAAAElFTkSuQmCC","orcid":"","institution":"Universidade Estadual de Maringá","correspondingAuthor":true,"prefix":"","firstName":"Brennda","middleName":"Ribeiro","lastName":"Paupitz","suffix":""},{"id":277284285,"identity":"5b4bf2d9-ffd0-453a-9cc0-0d0a6086414e","order_by":1,"name":"Pablo Américo Barbieri","email":"","orcid":"","institution":"Universidade Estadual de Maringá","correspondingAuthor":false,"prefix":"","firstName":"Pablo","middleName":"Américo","lastName":"Barbieri","suffix":""},{"id":277284286,"identity":"2acd35fb-fa05-4808-b546-000888bba755","order_by":2,"name":"Luara Lupepsa","email":"","orcid":"","institution":"Universidade Estadual de Maringá","correspondingAuthor":false,"prefix":"","firstName":"Luara","middleName":"","lastName":"Lupepsa","suffix":""},{"id":277284287,"identity":"398aeac2-9d41-450a-893c-3def99ea5a35","order_by":3,"name":"Carlos Alexandre Fernandes","email":"","orcid":"","institution":"Universidade Estadual de Maringá","correspondingAuthor":false,"prefix":"","firstName":"Carlos","middleName":"Alexandre","lastName":"Fernandes","suffix":""},{"id":277284288,"identity":"326d0488-3c46-4d97-8b7f-3c204d3aa6c9","order_by":4,"name":"Ana Luiza Brito Portela-Castro","email":"","orcid":"","institution":"Universidade Estadual de Maringá","correspondingAuthor":false,"prefix":"","firstName":"Ana","middleName":"Luiza Brito","lastName":"Portela-Castro","suffix":""},{"id":277284289,"identity":"bdc266fa-63d7-41f3-8140-1c62fdb6508b","order_by":5,"name":"Luciana Andreia Borin-Carvalho","email":"","orcid":"","institution":"Universidade Estadual de Maringá","correspondingAuthor":false,"prefix":"","firstName":"Luciana","middleName":"Andreia","lastName":"Borin-Carvalho","suffix":""}],"badges":[],"createdAt":"2024-03-07 19:51:05","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4031547/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4031547/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":52538244,"identity":"499c97e8-73b3-40d5-b333-ec0817d7fd90","added_by":"auto","created_at":"2024-03-12 16:55:31","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":6911414,"visible":true,"origin":"","legend":"\u003cp\u003ePhotomicrograph of \u003cem\u003eA. lacustris\u003c/em\u003eliver chronically exposed to Metformin. (A) Control; (B) 50 µg/L concentration; (C) 100 µg/L concentration; (D) 1000 µg/L concentration; (E) 10,000 µg/L concentration. Arrows indicate cytoplasmic vacuolization in hepatocytes. Arrowheads indicate the presence of proteinaceous fluid within blood vessels. Only in treated animals is a diffuse aspect of hydropic hepatocytes observed.\u003c/p\u003e","description":"","filename":"OnlineFigure11.png","url":"https://assets-eu.researchsquare.com/files/rs-4031547/v1/c9fdb08f0cef9a74447cf4f3.png"},{"id":52538242,"identity":"2bbe35e7-bca7-43ba-877c-d37b885a27d1","added_by":"auto","created_at":"2024-03-12 16:55:31","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":682800,"visible":true,"origin":"","legend":"\u003cp\u003eGraph depicting a comparison of treated groups with the control group. (a) Hepatocyte count, (b) Cytoplasmic morphometric analysis, and (c) Nuclear morphometric analysis. * - Significant difference compared to control (p \u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"OnlineFigure21.png","url":"https://assets-eu.researchsquare.com/files/rs-4031547/v1/bbd5fd7bb5fe7ddcf340b3fd.png"},{"id":54409015,"identity":"0c52b953-5266-40c0-9473-e5183b800351","added_by":"auto","created_at":"2024-04-10 04:55:33","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2327477,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4031547/v1/5ce10e19-1852-4450-b4a8-6b363145064b.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Environmental concentrations of the antidiabetic Metformin cause liver damage in Astyanax lacustris (Lütken, 1875) individuals after chronic exposure","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eWastewater treatment plants have been the focus of studies regarding introducing pollutants, such as pharmaceutically active compounds, into the environment (Ghirardini et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Even in non-therapeutic doses, these compounds can still interfere with the environment and human health (Daughton and Ruhoy, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). In general, these studies range from evaluating the concentration of medications in the environment to removal alternatives, which often involve the use of bioreactors (Ghirardini et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Goswami et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Scheurer et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2012\u003c/span\u003e) aiming to minimize the damages caused by these pollutants (Vatovec; Wagoner; Evans, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2017\u003c/span\u003e).\u003c/p\u003e \u003cp\u003ePharmaceuticals are released into the environment through two main routes. Improper disposal of medications that will no longer be used is quite common, whether due to leftover medications, their expiration, or patient non-adherence to treatment, which usually leads to the disposal of the drugs in the toilet or household trash. Another route is the excretion of the unmetabolized component through the patient's feces and urine or the release of topically used medication into bathwater (Daughton and Ruhoy, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). This disposal contributes to the pollution of groundwater and surface waters through septic systems and reaches rivers and lakes, the primary recipients (Carrara et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). Pharmaceuticals of high relevance are often found in low concentrations in the environment, and numerous knowledge gaps persist, underscoring the imperative for further research to comprehensively evaluate the potential risks posed by pharmaceuticals in aquatic ecosystems (Fent; Weston; Caminada, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2006\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAmong these pharmaceuticals, the antidiabetic Metformin (1,1-dimethylbiguanide), which is a first-line treatment for patients with type 2 diabetes mellitus (Scheurer; Sacher; Brauch, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Idf, 2017), has gained significant attention. Because it is widely used worldwide and is not metabolized by the human body, it is found in its non-biotransformed form in aquatic ecosystems across diverse regions, as shown by studies (Kane, 2020; Graham et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Trautwein and K\u0026uuml;mmerer (\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2011\u003c/span\u003e) and Scheurer et al. (\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2012\u003c/span\u003e) demonstrated through biodegradation tests with microorganisms that Metformin is a drug not readily degraded in the aquatic environment. Besides, conventional wastewater treatment is inefficient in eliminating this compound (Carbuloni et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2020\u003c/span\u003e)\u003c/p\u003e \u003cp\u003eIn the natural aquatic environment, fish interact with biological systems and substances such as pharmaceuticals, which trigger physiological changes. Environmental pollution alters the chemical composition of natural habitats, affecting organisms that are not the target of these compounds' action (Monteiro et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). Adverse effects of the antidiabetic have been observed in some fish species, such as \u003cem\u003ePimephales promelas\u003c/em\u003e, \u003cem\u003eOryzias latipes\u003c/em\u003e, \u003cem\u003eBetta splendens\u003c/em\u003e, and \u003cem\u003eAstyanax lacustris\u003c/em\u003e. The observed changes include variations in vitellogenin levels, estrogen receptor levels, gonadotropin-releasing hormone levels, alterations in the expression of the CYP19A gene, presentation of oxidative stress, endocrine disruption, effects on animal behavior and morphological modifications in gills (Crago et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Lee et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Maclaren; Wisniewski; Maclaren, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Barbieri, 2022). Among the most notable effects of Metformin is the occurrence of intersex individuals in male \u003cem\u003eP. promelas\u003c/em\u003e, induced by activation of vitellogenin mRNA transcription, a protein responsible for vitellogenesis that usually is only expressed in females (Niemuth et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2015\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe impact of xenobiotics, such as Metformin, on these organisms can be assessed by analyzing biochemical disturbances caused, allowing fish to be used as bioindicators in polluted environments (Monteiro et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). The liver plays an essential role in the metabolism of xenobiotics, making it susceptible to damage from these agents as hepatic tissue is vulnerable to environmental changes, often serving as a biomarker for assessing damage (Monteiro et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2006\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cem\u003eAstyanax lacustris\u003c/em\u003e is considered an excellent species for experimental modeling due to its sensitivity and response to the presence of contaminants in the environment, often used as a bioindicator species (Sposito et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). It is widely distributed in Brazilian watersheds and holds significant ecological importance (Lucena, Soares, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Vilella, Becker, Hartz, 2002). Based on this, the present study aimed to evaluate the potential ecotoxicological impacts of Metformin on the liver of \u003cem\u003eA. lacustris\u003c/em\u003e fish specimens, a species with wide distribution in the neotropical region and considerable ecological significance due to its interactions with other ecosystem organisms.\u003c/p\u003e"},{"header":"2. Material and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. \u003cem\u003eFish Acquisition and Maintenance\u003c/em\u003e\u003c/h2\u003e \u003cp\u003e \u003cem\u003eA. lacustris\u003c/em\u003e individuals were obtained from commercial establishments from fish farms where the animals did not come into contact with effluents. Transportation was carried out using plastic bags containing 1/3 water and 2/3 oxygen, placed in a styrofoam box to protect against shocks and reduce fish stress. The specimens were kept in the fish vivarium located in block T-10, where they were maintained in water tanks with 150 liters containing 100 liters of water at ambient temperature and lighting conditions. They were fed with commercial feed for small fish once a day until the experimental procedures. Throughout the experiments, parameters such as water temperature, oxygen content, conductivity, and pH were consistently monitored and maintained within the optimal range for these fish, ensuring that these factors did not influence the results.\u003c/p\u003e \u003cp\u003e The main parameters and variables required for the well-being of the animals, the stocking density, and the water exchange in the aquariums were carried out according to CONCEA Normative Resolution No. 34, dated July 27, 2017.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. \u003cem\u003ePreparation of Metformin Stock Solution\u003c/em\u003e\u003c/h2\u003e \u003cp\u003eFor the experiments, two sources of Metformin were used: metformin sigma P.A (used for animals exposed to concentrations in the microgram range) and commercial Medley metformin (used for animals exposed to milligram concentrations).\u003c/p\u003e \u003cp\u003eA stock solution with a concentration of 7800 mg/L was prepared from commercial Medley metformin (metformin hydrochloride 780 mg\u0026thinsp;+\u0026thinsp;excipients q.s.p). The tablets were crushed using a mortar and pestle and diluted in distilled water. A stock solution with a concentration of 50 \u0026micro;g/L was prepared from metformin sigma P.A. Both solutions were aliquoted and stored in a refrigerated and dark location.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3. \u003cem\u003eMetformin Exposure\u003c/em\u003e\u003c/h2\u003e \u003cp\u003eJuvenile male animals (30 to 40 days post-hatch) were randomly selected and exposed for 90 days. The group included a control subgroup and four additional subgroups based on the concentration of Metformin: 50 \u0026micro;g/L, 100 \u0026micro;g/L, 1000 \u0026micro;g/L, and 10,000 \u0026micro;g/L (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The distribution of fishes among the concentration groups was carried out following the blinding methods. The concentrations of 50 \u0026micro;g/L and 100 \u0026micro;g/L were established based on concentrations found in surface waters of different aquatic environments, using values close to the minimum and maximum (Scheurer et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Scheurer et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). The concentration of 10,000 \u0026micro;g/L corresponds to the NOEC value for zebrafish in chronic tests (Moermond and Smit, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). The 1 mg/L concentration was used to test a concentration ten times lower than the NOEC.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eGroup 1: Juvenile fish aged 30 to 40 days post-hatching. Chronic treatment (90 days): 5 animals for each concentration\u0026thinsp;+\u0026thinsp;control.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colspan=\"6\" nameend=\"c7\" namest=\"c2\"\u003e \u003cp\u003eSubgroups\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGroup\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eTotal\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eConcentration\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e50 ug/L\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e100 ug/L\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1000 \u0026micro;g/L\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e10.000 \u0026micro;g/L\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eControle\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAnimals\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e25\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4. \u003cem\u003eEuthanasia Method for Animals\u003c/em\u003e\u003c/h2\u003e \u003cp\u003eEach animal was immersed in a clove oil/eugenol solution after being removed from the aquarium, following the instructions of Inoue and Morais (2007). The anesthetic solution was prepared: 1 ml of the anesthetic (eugenol) diluted in 3 ml of alcohol. Subsequently, 0.5 ml of this solution was added to each 1 liter of water. The fish were handled only after no reaction to any physical stimulus, indicating death by anesthetic overdose. The ethics committee (CEUA/UEM) approved the animal experimentation protocol under number 6409140218.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5. \u003cem\u003eHistological Analysis\u003c/em\u003e\u003c/h2\u003e \u003cp\u003eLiver samples were washed in 0.9% saline solution and fixed in aqueous Bouin's solution for 12 hours (Behmer et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e1976\u003c/span\u003e), then stored in 70% alcohol. The material was dehydrated in increasing series of alcohols (80%, 90%, and 100%), cleared in xylene, and embedded in paraffin for histological processing. Semi-serial transverse sections of 5\u0026micro;m thickness were obtained using a LEICA rotary microtome at the Animal Histotechnique Laboratory of the Department of Morphological Sciences at Universidade Estadual de Maring\u0026aacute;. Slides were stained using the Hematoxylin-Eosin (H.E.) method (Behmer et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e1976\u003c/span\u003e). The light microscope (Olympus CX31RBSFA) was used to verify morphological analysis and liver alterations. All analyses were conducted using the experimental blinding method, where it was not possible to know which group the analyzed fish belonged to. Additionally, the samples were analyzed by two different researchers to ensure the same result.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6. \u003cem\u003eQuantitative Analysis of Hepatic Cell Numbers and Morphometric Analysis of Hepatocyte Cytoplasm and Nucleus\u003c/em\u003e\u003c/h2\u003e \u003cp\u003eImages of H.E.-stained sections were captured using an Olympus BX41\u0026reg; optical microscope (40x objective) with an Olympus Q-Color\u0026reg; 3 camera, connected to a computer with Q-Capture\u0026reg; software.\u003c/p\u003e \u003cp\u003eIn the hepatocyte counting analysis, 50 images/animal were measured, totaling 250 images/group. For morphometry, 200 hepatocytes/animal were measured, totaling 1000 hepatocytes/group. The Image Pro-Plus\u0026reg; 4.5 software (Media Cybernetics) was used for morphometric analyses.\u003c/p\u003e \u003cp\u003eThe obtained data were checked for normality using the Kolmogorov-Smirnov test. Subsequently, they were subjected to a one-way analysis of variance (ANOVA) followed by Tukey's post-test. The significance level adopted was 5%, and the results were expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error. The statistical program used was GraphPad Prism version 8.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cp\u003eMorphologically, the livers of the control animals of \u003cem\u003eA. lacustris\u003c/em\u003e displayed polygonal hepatocytes with cytoplasmic vacuolation, centrally located nuclei with evident nucleoli (Fig.\u0026nbsp;1a) similar to what has been described for other characids (Petcoff et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Marcon et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2015\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe vacuoles appeared typically flocculent, angular with smooth edges, and minimal displacement of the nucleus, characteristic of vacuoles that store glycogen.\u003c/p\u003e \u003cp\u003eIn control animals, an eosinophilic staining of the hepatocellular cytoplasm was observed (Fig.\u0026nbsp;2A), whereas in animals exposed to Metformin, increased basophilia was noted in the hepatocyte cytoplasm (Figs.\u0026nbsp;2B, 2C, 2D). In animals exposed to the drug, it was also possible to observe hydropic hepatocytes, characterized by diffuse cytoplasmic clearing, maintenance of the nucleus in a central position, and architectural distortion of hepatocyte cords with compression of the sinusoids. Another aspect noticed was increased eosinophilic fluid in several blood vessels (Figs.\u0026nbsp;2B, 2C, 2D - arrowhead).\u003c/p\u003e \u003cp\u003e \u003cb\u003eFigure 1 -\u003c/b\u003e Photomicrograph of \u003cem\u003eA. lacustris\u003c/em\u003e liver chronically exposed to Metformin. (A) Control; (B) 50 \u0026micro;g/L concentration; (C) 100 \u0026micro;g/L concentration; (D) 1000 \u0026micro;g/L concentration; (E) 10,000 \u0026micro;g/L concentration. Arrows indicate cytoplasmic vacuolization in hepatocytes. Arrowheads indicate the presence of proteinaceous fluid within blood vessels. Only in treated animals is a diffuse aspect of hydropic hepatocytes observed.\u003c/p\u003e \u003cp\u003eThere was a decrease in the number of these cells compared to the control-exposed individuals (Fig.\u0026nbsp;1b and 2a). The animals in the control group had an average of 150 hepatocytes per field/image, while at the concentration of 50 \u0026micro;g/L, this value dropped to an average of 125 hepatocytes per field/image. At concentrations of 100 \u0026micro;g/L, 1000 \u0026micro;g/L, and 10,000 \u0026micro;g/L, the cell count was even lower, with an average of 80 hepatocytes per field/image. Statistically, the data from all concentrations showed a significant effect (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05) compared to the control.\u003c/p\u003e \u003cp\u003eBased on the cell count result, morphometry of the cytoplasmic area of the cells was performed to corroborate the previous analysis. Compared to the control, there was a progressive increase in cytoplasm size according to the metformin concentration (Fig.\u0026nbsp;1B), demonstrating a dose-dependent relationship. Statistically, the results showed a significant effect (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003eAs a result, the nuclear area of the hepatocytes was measured, revealing a progressive and significant increase (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05) in nucleus size according to the dose increase, as observed with the cytoplasm (Fig.\u0026nbsp;1C). The concentration that exhibited the largest nuclear area was 10,000 \u0026micro;g/L, corresponding to the NOEC concentration.\u003c/p\u003e \u003cp\u003e \u003cb\u003eFigure 2 -\u003c/b\u003e Graph depicting a comparison of treated groups with the control group. (a) Hepatocyte count, (b) Cytoplasmic morphometric analysis, and (c) Nuclear morphometric analysis. * - Significant difference compared to control (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003eVacuolization was found in the cytoplasm of hepatocytes in the control animals, and it was more pronounced in the hepatocytes of fish exposed to Metformin (Fig.\u0026nbsp;2).\u003c/p\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eThis study conducted a quantitative and qualitative analysis of the structural parameters of hepatocytes in the liver of an experimental model exposed to Metformin, A. lacustris, revealing alterations such as decreased cell count, progressive cytoplasmic and nuclear enlargement, and demonstrating dose dependence. Furthermore, other significant changes occurred in liver cells, including vacuole size, increased basophilia in the cytoplasm, architectural distortion of hepatocyte cords with sinusoidal compression, and increased eosinophilic fluid content in various blood vessels. These findings indicate profound histochemical alterations in the hepatocytes of these animals. While the physiological and structural liver structure is highly conserved among vertebrates, the liver of teleost fish is susceptible to environmental changes, whether in natural ecosystems or laboratory exposures, exhibiting both morphological and physiological changes in response to contamination (Wolf and Wheeler, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Santos et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Histopathological analysis is often required to assess and determine liver damage presence or absence under toxicological evaluation (Wolf and Wolfe, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2005\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eOur results demonstrate that Metformin, a significant emerging contaminant in wastewater and surface waters, significantly impacts the liver of \u003cem\u003eA. lacustris\u003c/em\u003e at environmentally relevant concentrations. Metformin, based on the PEC/PNEC value, has been considered a low environmental risk drug (Caldwell et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), where PEC is the predicted environmental concentration for a specific environment, and PNEC is the predicted no-effect concentration, below which adverse effects are unlikely to occur during exposure. The PNEC for chronic toxicity is typically estimated using the NOEC - no observed effect concentration (Johnson et al., 2009). The established NOEC value for Metformin is 10 mg/L for \u003cem\u003eDanio rerio\u003c/em\u003e, and PNEC is 1 mg/L (Caldwell et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), meaning that this value corresponds to the highest concentration of the substance that does not cause statistically significant adverse effects in organisms under the test exposure time and conditions.\u003c/p\u003e \u003cp\u003eHowever, even at concentrations below the described PNEC value for Metformin, the results of this study demonstrate the occurrence of histopathological alterations in the liver. Moreover, a dose-dependent trend was observed in the reported morphometric effects, as higher concentrations of the compound in the water led to more significant results. Based on our data, we can conclude that a chemical substance does not cause death in aquatic organisms in toxicity tests does not indicate that it does not cause pathological damage to them.\u003c/p\u003e \u003cp\u003eThe alteration from eosinophilic staining of the hepatocellular cytoplasm in control animals to increased basophilia in treated animals observed in this study may be correlated with vitellogenin production, a protein responsible for oocyte development in females. According to the OECD document guiding diagnosing histopathological changes in fish exposed to potential endocrine disruptors (Johnson et al., 2009), a diffuse increase in hepatocellular basophilia is observed in animals exposed to a compound with estrogenic activity. This increase in basophilia, associated with increased vitellogenin production, presumably mimics the expanded metabolic state (e.g., increased endoplasmic reticulum) required for vitellogenin production in reproductively active females.\u003c/p\u003e \u003cp\u003eVan den Belt-K et al. (2002) also detected hepatocellular basophilia in female and male zebrafish exposed to an estrogen analog (ethinyl estradiol), indicating a high mRNA content. This hepatocyte basophilia coincides with high plasma vitellogenin levels, showing active vitellogenin synthesis in the liver. According to the authors, in exposed males but not in control animals, capillaries (and other vessels) are dilated and filled with eosinophilic plasma, indicative of high protein content (vitellogenin). Similarly, our results also showed an increase in the amount of eosinophilic protein fluid in various blood vessels.\u003c/p\u003e \u003cp\u003eThe increase in intracellular fluid, characterizing hydropic hepatocytes, also known as hydropic degeneration or cellular edema, is the first manifestation of nearly all forms of cellular damage. It is a reversible, non-lethal alteration. Hydropic degeneration is the accumulation of water within the intracellular environment, resulting from imbalances in osmotic gradient control at the level of the cytoplasmic membrane and mechanisms of absorption, elimination of water, and intracellular electrolytes. Intracellular water accumulation occurs due to the cell's inability to maintain ionic balance and fluid homeostasis, resulting from failures in energy-dependent pumps of cell membranes (Miller and Zachary, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). The correlation between Metformin and decreased ATP production, leading to the losses, occurs due to the inhibition of Complex I of the mitochondrial respiratory chain (Faure et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Consequently, the decline in intracellular ATP could interfere with ion transport through the Na+/K+-ATPase and thus disrupt the osmotic balance of hepatocytes in animals exposed to Metformin, explaining the observation of hydropic hepatocytes.\u003c/p\u003e \u003cp\u003eAnalyzing \u003cem\u003eA. lacustris\u003c/em\u003e exposed to Metformin, previously conducted by our team, also revealed cellular changes associated with decreased intracellular ATP, such as the presence of echinocytes seen in blood smears under light microscopy and loss of microdigitations observed under scanning electron microscopy in gills, both caused by cytoskeleton modulations involving dissociations between spectrin-actin, which require energy for stability.\u003c/p\u003e \u003cp\u003eThe morphometric parameters of the cytoplasm and nucleus showed increased volume with increasing metformin dose. This growth reflects a response found by Mac\u0026ecirc;do et al. (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) in \u003cem\u003eA. lacustris\u003c/em\u003e exposed to river waters from different regions containing various contaminants, mainly metals, demonstrating that exposure of this species to environmental pollutants results in this hepatic response. According to Wolf and Wolfe (\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2005\u003c/span\u003e), the increase in cytoplasmic volume found in hepatocytes can be caused by hypertrophy due to organelle proliferation (hypertrophy), failure in the mitosis process causing megalocytosis, and osmotic imbalance leading to increased intracellular water (hydropic degeneration or edema) and cytoplasmic vacuolization. Based on the characteristics observed in \u003cem\u003eA. lacustris\u003c/em\u003e hepatocytes, it is assumed that the increased cell volume occurred due to hydropic degeneration and glycogen accumulation in the vacuoles. Hepatic glycogen content increased in \u003cem\u003eSalmo trutta f. fario\u003c/em\u003e exposed to environmentally relevant concentrations of Metformin (Jacob et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2018\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eBased on the increase in cytoplasmic and nuclear volume with increasing antidiabetic concentration, it is presumed that the observed decrease in cell count per field/image occurred due to increased cell volume rather than a decrease in hepatocyte number. However, the hepatosomatic index should be analyzed to confirm this hypothesis.\u003c/p\u003e \u003cp\u003eIn addition to the increase in cytoplasmic volume, there was an increase in nuclear size compared to the control subgroup. In the work of Santos et al. (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2013\u003c/span\u003e), brown trout individuals, also teleosts, exposed to a hepatocarcinogenic initiator, N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), exhibited hepatocytes with a larger nuclear volume than \"normal\" hepatocytes, indicating that the nucleus of the healthy hepatocyte differs structurally and functionally from that of the exposed hepatocyte, similar to our results. The increase in nuclear volume observed here may be associated with increased expression of genes related to protein synthesis, as studies have already shown overexpression of genes used as markers of damage caused by endocrine disruptors. The egg yolk protein, vitellogenin (VTG), consists of a polypeptide whose increased levels in male fish have been attributed to hepatotoxic responses mediated by contaminants lacking hormonal structure but with potential endocrine disruptor effects, serving as an essential biomarker (Baumann et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Niemuth et al. (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2015\u003c/span\u003e) found in their study with \u003cem\u003eP. promelas\u003c/em\u003e exposed to environmentally relevant concentrations of Metformin for 28 days that there was overexpression of VTG mRNA in males of the species, while levels in exposed females did not differ from the control. Although insignificant, Plasma VTG levels in males were higher than in the control. The increase in VTG mRNA expression was also observed in male \u003cem\u003eP. promelas\u003c/em\u003e exposed to another contaminant, the synthetic estrogen 17α-ethinylestradiol (EE2) (Lattier et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2002\u003c/span\u003e).\u003c/p\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003eWe observed that \u003cem\u003eA. lacustris\u003c/em\u003e individuals exposed to different concentrations of metformin, including environmentally relevant concentrations, exhibited significant alterations in liver tissue compared to the control group. This research demonstrates the environmental relevance of metformin as a contaminant. Due to its widespread use worldwide and persistence in the aquatic ecosystem, this compound needs to be studied as a potential emerging environmental contaminant, considering the obtained results here.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eCONFLICT OF INTEREST\u003c/h2\u003e \u003cp\u003eAuthors declare that they do not have any conflict of interest.\u003c/p\u003e \u003ch2\u003eEthics approval\u003c/h2\u003e \u003cp\u003e Ethical approval for this study was obtained from Ethics Committee on the Use of Animals (CEUA - State University of Maring\u0026aacute;, Paran\u0026aacute;, Brazil) under decision number 6409140218.\u003c/p\u003e\u003ch2\u003eFUNDING INFORMATION\u003c/h2\u003e \u003cp\u003eThis study was financed by CAPES (Coordenadoria de Aperfei\u0026ccedil;oamento de Ensino Superior).\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eAll authors discussed the results and contributed to the final manuscript. Luciana Andreia Borin-Carvalho, Brennda Ribeiro Paupitz and Ana Luiza de Brito Portela-Castro conceived of the presented idea. Material preparation, data collection and analysis were performed by Brennda Ribeiro Paupitz, Luara Lupepsa and Pablo Americo Barbieri. The manuscript was written by Brennda Ribeiro Paupitz with support from Luciana Andreia Borin-Carvalho, Ana Luiza de Brito Portela-Castro. All authors read and approved the final manuscript.\u003c/p\u003e\u003ch2\u003eACKNOWLEDGEMENTS\u003c/h2\u003e \u003cp\u003eWe thank of the Centro de Microscopia (CMI) of Complexo de Centrais de Apoio \u0026agrave; pesquisa (COMCAP) at the Universidade Estadual de Maring\u0026aacute; (UEM), Maring\u0026aacute;, PR in processing material used and assistance in handling the equipment. This work was supported by the Coordena\u0026ccedil;\u0026atilde;o de Aperfei\u0026ccedil;oamento de Pessoal de N\u0026iacute;vel Superior (CAPES).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eBarbieri, P.A., Mari-Ribeiro, IP, Lupepsa, L, Gigliolli, AAS, Paupitz, BR, Melo, RF; Mello, EVSL, Portela-Castro, ALB, Borin-Carvalho, LA, 2022. Metformin-induced alterations in gills of the freshwater fish \u003cem\u003eAstyanax lacustris\u003c/em\u003e (L\u0026uuml;tken, 1875) detected by histological and scanning electron microscopy. Ecotoxicology 31, 1205\u0026ndash;1216. https://doi.org/10.1007/s10646-022-02580-0\u003c/li\u003e\n\u003cli\u003eBaumann, L., Holbech, H, Schmidt-Posthaus, H, Moissl, AP, Hennies, M, Tiedemann, J, Weltje, L, Segner, H, Braunbeck, T, 2020. Does hepatotoxicity interfere with endocrine activity in zebrafish (\u003cem\u003eDanio rerio\u003c/em\u003e)? 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A critical review of histopathological findings associated with endocrine and non-endocrine hepatic toxicity in fish models. Aquatic Toxicology, 197, 60-78. Doi: 10.1016/j.aquatox.2018.01.013\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Metformin, liver, hepatocyte, endocrine disruptor, aquatic pollution","lastPublishedDoi":"10.21203/rs.3.rs-4031547/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4031547/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eDrugs are emerging contaminants that provide concern when it comes to the adverse effects they can cause on organisms that are not the target of therapeutic action. Because water treatment methods do not entirely remove them, they are found in worrying concentrations in the aquatic environment. Antidiabetic Metformin has been found in the environment worldwide, and studies show it has a potential endocrine disrupting effect. However, more research is needed regarding its impact on bioindicator organisms, such as fish. This work aimed to evaluate the effect of chronic exposure to Metformin in the liver, the organ responsible for the metabolism of xenobiotics, from Astyanax lacustris. The results obtained from histological sections of the organ show that Metformin induced liver damage since the number, size, and composition of hepatocytes have changed. This study demonstrates the need for more research on the damage metformin can cause aquatic life.\u003c/p\u003e","manuscriptTitle":"Environmental concentrations of the antidiabetic Metformin cause liver damage in Astyanax lacustris (Lütken, 1875) individuals after chronic exposure","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-03-12 16:55:26","doi":"10.21203/rs.3.rs-4031547/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"1e3bb81c-8ae3-49ef-889d-d99777ed90e8","owner":[],"postedDate":"March 12th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-04-10T04:47:25+00:00","versionOfRecord":[],"versionCreatedAt":"2024-03-12 16:55:26","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4031547","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4031547","identity":"rs-4031547","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}