Erythrocyte alterations in specimens of Danio rerio caused by exposure to metformin.

The antidiabetic drug metformin is widely prescribed around the world. However, its permanence in different environmental concentrations has been associated with adverse toxicological effects in organisms that do not target its therapeutic action. In the aquatic environment, fish such as the Zebrafish (Danio rerio) have been considered potential bioindicators of environmental impacts and used as experimental models in toxicological studies due to the sensitivity of these species to different types of contaminants, including pharmaceuticals. Thus, this study aimed to analyze metformin's cytotoxic effects on Danio rerio erythrocytes. The animals were submitted to different concentrations of the drug (50 µg/L, 100 µg/L, 150 µg/L, and 10000 µg/L) for 365 days and subsequently observed employing light microscopy and scanning electron microscopy (SEM) to evaluate the alterations that occurred. Exposure of animals to metformin led to significant erythrocyte cell abnormalities across all tested concentrations, with a particularly pronounced effect at the higher concentration previously defined as the NOEC (No Observed Effect Concentration). Remarkable abnormalities included cytoplasmic vacuoles, echinocytes, and vesicle-like cytoplasmic fragments. These findings suggest that metformin, even at concentrations similar to those found in nature and at the NOEC level, exhibits cytotoxic potential in D. rerio, raising concerns about its potential health impacts.

Network Toxicology Prediction and Molecular Docking-based Strategy to Explore the Potential Toxicity Mechanism of Metformin Chlorination Byproducts in Drinking Water.

BACKGROUND: Metformin (MET), a worldwide used drug for treating type 2 diabetes but not metabolized by humans, has been found with the largest amount in the aquatic environment. Two MET chlorination byproducts, including Y and C, were transformed into drinking water during chlorination. However, the potential toxicity of the byproducts in hepatotoxicity and reproduction toxicity remains unclear. METHODS: The TOPKAT database predicted the toxicological properties of metformin disinfection by-products. The targets of metformin disinfection by-products were mainly obtained from the PharmMapper database, and then the targets of hepatotoxicity and reproductive toxicity were screened from GeneCards. The overlapping targets of toxic component targets and the hepatotoxicity or reproduction toxicity targets were regarded as the key targets. Then, the STRING database analyzed the key target to construct a protein-protein interaction network (PPI) and GO, and KEGG analysis was performed by the DAVID platform. Meanwhile, the PPI network and compound- target network were constructed by Cytoscape 3.9.1. Finally, Discovery Studio 2019 software was used for molecular docking verification of the two toxic compounds and the core genes. RESULTS: Y and C exhibited hepatotoxicity, carcinogenicity, and mutagenicity evaluated by TOPKAT. There were 22 potential targets relating to compound Y and hepatotoxicity and reproduction toxicity and 14 potential targets relating to compound C and hepatotoxicity and reproduction toxicity. PPI network analysis showed that SRC, MAPK14, F2, PTPN1, IL2, MMP3, HRAS, and RARA might be the key targets; the KEGG analysis indicated that compounds Y and C caused hepatotoxicity through Hepatitis B, Pathways in cancer, Chemical carcinogenesis-reactive oxygen species, Epstein-Barr virus infection; compound Y and C caused reproduction toxicity through GnRH signaling pathway, Endocrine resistance, Prostate cancer, Progesterone-mediated oocyte maturation. Molecular docking results showed that 2 compounds could fit in the binding pocket of the 7 hub genes. CONCLUSION: This study preliminarily revealed the potential toxicity and possible toxicity mechanism of metformin disinfection by-products and provided a new idea for follow-up research.

Metformin exposure altered intestinal microbiota composition and metabolites in amphibian larvae.

The antidiabetic pharmaceutical metformin (MET) is largely unmetabolized by the human body. Its residues are readily detectable in various aquatic environments and may have adverse impacts on the growth and survival of aquatic species. To date, its toxicological effects have scarcely been explored in non-fish species. Here, we exposed the tadpoles of black-spotted pond frog (Pelophylax nigromaculatus) to different concentrations (0, 1, 10 and 100 µg/L) of MET for 30 days and measured the body size, intestinal microbiota and metabolites to evaluate potential effects of MET exposure in amphibian larvae. MET exposure did not affect the growth and intestinal microbial diversity of tadpoles. However, intestinal microbial composition changed significantly, with some pathogenic bacteria (e.g., bacterial genera Salmonella, Comamonas, Stenotrophomonas, Trichococcus) increasing and some beneficial bacteria (e.g., Blautia, Prevotella) decreasing in MET-exposed tadpoles. The levels of some intestinal metabolites associated with growth and immune performance also changed significantly following MET exposure. Overall, our results indicated that exposure to MET, even at environmentally relevant concentrations, would cause intestinal microbiota dysbiosis and metabolite alteration, thereby influencing the health status of non-target aquatic organisms, such as amphibians.

Health impact assessment after Danio rerio long-term exposure to environmentally relevant concentrations of metformin and guanylurea.

The antidiabetic drug metformin (MET) and its metabolite guanylurea (GUA) have been frequently and ubiquitously detected in surface water. Consequently, there has been a consistent rise in studying the toxicity of MET and GUA in fish over the past decade. Nonetheless, it is noteworthy that no study has assessed the harmful effects both compounds might trigger on fish blood and organs after chronic exposure. Taking into consideration the data above, our research strived to accomplish two primary objectives: Firstly, to assess the effect of comparable concentrations of MET and GUA (1, 40, 100 µg/L) on the liver, gills, gut, and brain of Danio rerio after six months of flow-through exposure. Secondly, to compare the outcomes to identify which compound prompts more significant oxidative stress and apoptosis in organs and blood parameter alterations. Herein, findings indicate that both compounds induced oxidative damage and increased the expression of genes associated with apoptosis (bax, bcl2, p53, and casp3). Chronic exposure to MET and GUA also generated fluctuations in glucose, creatinine, phosphorus, liver enzymes, red and white blood count, hemoglobin, and hematocrit levels. The observed biochemical changes indicate that MET and GUA are responsible for inducing hepatic damage in fish, whereas hematological alterations suggest that both compounds cause anemia. Considering GUA altered to a more considerable extent the values of all endpoints compared to the control group, it is suggested transformation product GUA is more toxic than MET. Moreover, based on the above evidence, it can be inferred that a six-month exposure to MET and GUA can impair REDOX status and generate apoptosis in fish, adversely affecting their essential organs' functioning.

Toxicological effects and underlying mechanisms of chlorination-derived metformin byproducts in Escherichia coli.

Chlorination-derived byproducts of the emerging contaminant metformin, such as (3E)-3-(chloroimino)-N,N-dimethyl-3H-1,2,4-triazol-5-amine (3,3-CDTA) and N-cyano-N,N-dimethylcarbaminmidic chloride (NCDC), occur in global waters and are toxic to organisms, from bacteria to mice. However, the mechanisms underlying their toxicity remain unknown. Here, we explored the toxicological effects and potential molecular mechanisms of 3,3-CDTA and NCDC at milligram concentrations, using Escherichia coli as a model organism. Compared with metformin (>300 mg/L), 3,3-CDTA and NCDC exerted stronger toxicity to E. coli, with a 4-h half maximal inhibitory concentration of 2.97 mg/L and 75.7 mg/L, respectively. Both byproducts disrupted E. coli cellular structures and components, decreased membrane potential and adenosine triphosphate (ATP) biosynthesis, and led to excessive reactive oxidative species (ROS), as well as the ROS-scavenging enzymes superoxide dismutase and catalase. Proteomic analysis and molecular docking supported these biomarker responses in the byproduct-treated E. coli, and indicated potential damage to DNA/RNA processes, while also provided novel insights into the toxicological and detoxified-byproduct effects at the proteome level. The toxicity-related events of NCDC and 3,3-CDTA included membrane disruption, oxidative stress, and abnormal protein expression. This study is the first to examine the toxicological effects of chlorination-derived metformin byproducts in E. coli and the associated pathways involved; thereby broadening our understanding regarding the toxicity and transformation risks of metformin throughout its entire life process.

Medicine designed to combat diseases of affluence affects the early development of fish. How do plastic microparticles contribute?

The incidence of diseases of affluence, such as diabetes mellitus, cardiovascular diseases, high blood pressure, and high cholesterol has been reported to rise. Consequently, the concentrations of residues of drugs designed to treat these diseases have been rising in water bodies. Moreover, the toxicity of these pharmaceuticals towards fish and other non-target organisms can be even enhanced by microplastic particles that are reportedly present in surface water. Therefore, the aim of this study was to describe the effects of three highly prescribed drugs, in particular metoprolol, enalapril, and metformin on fish early-life stages. Also, it was hypothesized that polystyrene microparticles will increase the toxicity of metoprolol to fish early-life stages. Embryonal acute toxicity tests on Danio rerio and Cyprinus carpio were carried out in order to describe the possible toxic effects of metoprolol, enalapril, and metformin. Also, the acute toxicity of polystyrene microparticles and the combination of metoprolol with polystyrene microparticles were tested on D. rerio embryos. Additionally, a 31-day long embryo-larval subchronic toxicity test was carried out with C. carpio in order to describe the long-term effects of low concentrations of metoprolol. The results of the study show that both metoprolol and enalapril have the potential to disrupt the early development of the heart in the embryonal stages of fish. Also, enalapril and metformin together with polystyrene microparticles seem to possibly disrupt the reproduction cycle and act as endocrine disruptors. Both pure polystyrene microparticles and the combination of them with metoprolol affect inflammatory processes in organisms. Additionally, metformin alters several metabolism pathways in fish early-life stages. The results of the study bring new evidence that even low, environmentally-relevant concentrations of pharmaceuticals have the potential to disrupt the early development of fish, particularly on a molecular level.

The elevated toxicity of the biodegradation product (guanylurea) from metformin and the antagonistic pattern recognition of combined toxicity: Insight from the pharmaceutical risk assessment and the simulated wastewater treatment.

The emerging contaminants metformin (MET) and its degradation product guanylurea (GUA) are released into aquatic environments through wastewater treatment plants (WWTPs). Thus, the environmental risks of wastewater with more treatments may be underestimated due to the lower effect concentration of GUA and the higher detected concentration of GUA in treated wastewater in comparison with MET. In this study, we aimed to investigate the combined toxicity mode of MET and GUA to Brachionus calyciflorus by simulating the degrees of wastewater treatments through adjustments to the ratio of MET and GUA in medium. The results showed that the 24 h-LC50 of MET, GUA, their mixtures of equal concentrations and the mixtures of equal toxic units to B. calyciflorus were 907.44, 544.53, 1185.82 and 940.52 mg/L, respectively, demonstrating GUA is significantly more toxic than MET. An antagonistic interaction between MET and GUA was found in mixture toxicity assessments. Compared with the control, MET treatments only significantly affected the intrinsic rate of population increase of rotifers (rm), while all life-table parameters were significantly affected by GUA. In addition, at medium and high concentrations (120 and 600 µmol/L), the net reproductive rate (R0) and rm of rotifers under GUA were significantly lower than those under MET. Notably, increased proportion of GUA relative to MET in binary-mixture treatments resulted in increased survival risk and reduced fecundity of rotifers. Moreover, the responses of population dynamics to exposures of MET and GUA were mainly attributed to the reproduction of rotifer, indicating that an improved wastewater treatment process is necessary to protect aquatic ecosystems. The study highlights the importance of considering the combined toxicity of emerging contaminants and degradation product in environmental risk assessment, especially the unintentional transformations of parent compound in treated wastewater.

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

The antidiabetic drug Metformin (MET), one of the most prevalent pharmaceuticals in the environment, is currently detected in surface waters in the range of ng/L to low µg/L. As current knowledge regarding the long-term effects of environmentally relevant concentrations of MET in nontarget organisms is limited, the present study aimed at investigating the generational effects of MET, in concentrations ranging from 390 to 14 423 ng/L in the model organism Danio rerio (up to 9 mpf), including the effects on its nonexposed offspring (until 60 dpf). We integrate several apical end points, i.e., embryonic development, survival, growth, and reproduction, with qRT-PCR and RNA-seq analyses to provide additional insights into the mode of action of MET. Reproductive-related parameters in the first generation were particularly sensitive to MET. MET parental exposure impacted critical molecular processes involved in the metabolism of zebrafish males, which in turn affected steroid hormone biosynthesis and upregulated male vtg1 expression by 99.78- to 155.47-fold at 390 and 14 432 MET treatment, respectively, pointing to an estrogenic effect. These findings can potentially explain the significant decrease in the fertilization rate and the increase of unactivated eggs. Nonexposed offspring was also affected by parental MET exposure, impacting its survival and growth. Altogether, these results suggest that MET, at environmentally relevant concentrations, severely affects several biological processes in zebrafish, supporting the urgent need to revise the proposed Predicted No-Effect Concentration (PNEC) and the Environmental Quality Standard (EQS) for MET.

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

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

Chronic exposure to realistic concentrations of metformin prompts a neurotoxic response in Danio rerio adults.

Metformin (MET) is among the most consumed drugs around the world, and thus, it is considered the uppermost drug in mass discharged into water settings. Nonetheless, data about the deleterious consequences of MET on water organisms are still scarce and require further investigation. Herein, we aimed to establish whether or not chronic exposure to MET (1, 20, and 40 µg/L) may alter the swimming behavior and induce neurotoxicity in Danio rerio adults. After 4 months of exposure, MET-exposed fish exhibited less swimming activity when compared to control fish. Moreover, compared with the control group, MET significantly inhibited the activity of AChE and induced oxidative damage in the brain of fish. Concerning gene expression, MET significantly upregulated the expression of Nrf1, Nrf2, BAX, p53, BACE1, APP, PSEN1, and downregulated CASP3 and CASP9. Although MET did not overexpress the CASP3 gene, we saw a meaningful rise in the activity of this enzyme in the blood of fish exposed to MET compared to the control group, which we then confirmed by a high number of apoptotic cells in the TUNEL assay. Our findings demonstrate that chronic exposure to MET may impair fish swimming behavior, making them more vulnerable to predators.

Metformin-induced alterations in gills of the freshwater fish Astyanax lacustris (Lütken, 1875) detected by histological and scanning electron microscopy.

The antidiabetic drug metformin is widely prescribed and found in different concentrations in the environment around the world, raising concern about potential impacts on aquatic life. Analyses of the effects of exposure of biological models to aquatic contaminants are important for assessing pollution effects on fish health. The gills of fishes represent primary targets of disturbance by pollutants, mainly because of the large surface of the respiratory epithelium and the high perfusion rate, which both help the entry of pollutants into this tissue. In this context, the aim of this work was to use gill histological analyses biomarkers to evaluate the toxicity of metformin on aquatic environmental systems, by means of chronic exposure for 90 days of Astyanax lacustris (lambari), an ecologically important neotropical species that can be used as an environmental bioindicator. Histopathological analyses were performed using Light and Scanning Electron Microscopy. The main changes were lamellar fusion, telangiectasia hyperplasia and disappearance of microridges. The morphological changes observed possibly interfere with the gill physiology, indicating an unfavorable situation to the presence of metformin in the water, pointing to a concern that metformin may pose a risk to Astyanax lacustris and likely to other fish species, compromising the dynamics of the aquatic ecosystem as a whole. Graphical abstract.

Metformin disrupts Danio rerio metabolism at environmentally relevant concentrations: A full life-cycle study.

Metformin (MET), an anti-diabetic pharmaceutical of large-scale consumption, is increasingly detected in surface waters. However, current knowledge on the long-term effects of MET on non-target organisms is limited. The present study aimed to investigate the effects of MET in the model freshwater teleost Danio rerio, following a full life-cycle exposure to environmentally relevant concentrations (390 to 14 423 ng/L). Considering that the mode of action (MoA) of MET on non-target organisms remains underexplored and that MET may act through similar human pathways, i.e., lipid and energy metabolisms, biochemical markers were used to determine cholesterol and triglycerides levels, as well as mitochondrial complex I activity in zebrafish liver. Also, the hepatosomatic index as an indication of metabolic disruption, and the expression levels of genes involved in MET's putative MoA, i.e. acaca, acadm, cox5aa, idh3a, hmgcra, prkaa1, were determined, the last by qRT-PCR. A screening of mRNA transcripts, associated with lipid and energy metabolisms, and other signaling pathways potentially involved in MET-induced toxicity were also assessed using an exploratory RNA-seq analysis. The findings here reported indicate that MET significantly disrupted critical biochemical and molecular processes involved in zebrafish metabolism, such as cholesterol and fatty acid biosynthesis, mitochondrial electron transport chain and tricarboxylic acid cycle, concomitantly to changes on the hepatosomatic index. Likewise, MET impacted other relevant pathways mainly associated with cell cycle, DNA repair and steroid hormone biosynthesis, here reported for the first time in a non-target aquatic organism. Non-monotonic dose response curves were frequently detected in biochemical and qRT-PCR data, with higher effects observed at 390 and 2 929 ng/L MET treatments. Collectively, the results suggest that environmentally relevant concentrations of MET severely disrupt D. rerio metabolism and other important biological processes, supporting the need to revise the proposed environmental quality standard (EQS) and predicted no-effect concentration (PNEC) for MET.

Metformin as an emerging concern in wastewater: Occurrence, analysis and treatment methods.

Metformin is a wonder drug used as an anti-hypoglycemic medication; it is also used as a cancer suppression medicament. Metformin is a first line of drug choice used by doctors for patients with type 2 diabetes. It is used worldwide where the drug's application varies from an anti-hypoglycemic medication to cancer oppression and as a weight loss treatment drug. Due to its wide range of usage, metformin and its byproducts are found in waste water and receiving aquatic environment. This leads to the accumulation of metformin in living beings and the environment where excess concentration levels can lead to ailments such as lactic acidosis or vitamin B12 deficiency. This drug could become of future water treatment concerns with its tons of production per year and vast usage. As a result of continuous occurrence of metformin has demanded the need of implementing and adopting different strategies to save the aquatic systems and the exposure to metformin. This review discuss the various methods for the elimination of metformin from wastewater. Along with that, the properties, occurrence, and health and environmental impacts of metformin are addressed. The different analytical methods for the detection of metformin are also explained. The main findings are discussed with respect to the management of metformin as an emerging contaminants and the major recommendations are discussed to understand the major research gaps.

Prevalence, production, and ecotoxicity of chlorination-derived metformin byproducts in Chinese urban water systems.

The widely used antidiabetic drug metformin has become an emerging contaminant of water systems. In a prior study, we demonstrated the marked mammalian toxicity of the disinfection-derived byproducts (DBPs) Y (yellow, C4H6ClN5) and C (colorless, C4H6ClN3), and here assess the distribution, formation, and ecotoxicity of these in Chinese urban water systems. A national tap water assessment showed that metformin and C concentrations were higher in large, dense urban areas and surface water sources than in sparsely populated areas and groundwater sources. Water types' analysis clearly showed that C derived from chlorination of metformin-contaminated water (up to 4308.5 ng/L) circulated from domestic water (0.7-9.7 ng/L) via sewage (2.3 ng/L in effluent) to surface water (0.6-3.5 ng/L). Simulated disinfection and aqueous stability results systematically showed rapid formation and 24 h stability of both byproducts, indicating high exposure odds for water users. Both byproducts showed clear but distinct toxic effects on the growth (72 h IC50, 0.6 mg/L for Y and 4.4 mg/L for C) and photosynthesis of the microalgae Pseudokirchneriella subcapitata at milligram levels. Combinedly, our work reveals that metformin byproducts have been disseminated to urban water cycle and contaminated tap water, increasing potential toxic risk for drinking water. Its outcomes provide a preliminary reference for future studies on the environmental fate and ecotoxicological effects of unintended DBPs formed in the chlorination of metformin-contaminated water.

Cytogenetic effects of antidiabetic drug metformin.

Metformin (MET) is the first-choice antidiabetic drug for type 2 diabetes mellitus treatment. In this study, the genotoxic potential of MET was evaluated by using chromosome aberrations (CAs), sister chromatid exchanges (SCEs), and micronucleus (MN) assays in human peripheral lymphocytes as well as comet assay in isolated lymphocytes. Human lymphocytes were treated with different concentrations of MET (12.5, 25, 50, 75, 100, and 125 µg/mL) for 24 h and 48 h. A negative and a positive control (Mitomycin-C-MMC, 0.20 µg/mL, for CA, SCE, and MN tests; hydrogen peroxide-H2O2, 100 µM, for comet assay) were also maintained. MET significantly increased the frequency of CAs at 48 h exposure (except 12.5 µg/mL) compared to the negative control. MET increased SCEs/cells in both treatment periods (except 12.5 µg/mL at 24 h). MET only increased the frequency of MN at 125 µg/mL. While MET significantly increased the comet tail length (CTL) at four concentrations (25, 75, 100, and 125 µg/mL), it did not affect comet tail intensity (CTI) (except 125 µg/mL) and comet tail moment (CTM) at all the treatments. All these data showed that MET had a mild genotoxic effect, especially at a long treatment period and higher concentrations in human lymphocytes in vitro. However, further in vitro and especially in vivo studies should be conducted to understand the detailed genotoxic potential of MET.HighlightsMetformin increased the frequency of CAs and SCEs, especially at 48-h exposure time in human lymphocytes.This antidiabetic drug increased the frequency of MN only at the highest concentration tested (125 µg/mL).Metformin significantly increased the comet tail length in all treatments (except 50 µg/mL).The drug did not significantly affect the comet tail intensity (except 125 µg/mL) and comet tail moment in all treatments.

Metformin ameliorates acetaminophen-induced sub-acute toxicity via antioxidant property.

Acetaminophen or N-acetyl-p-amino-phenol (APAP) is a drug which is available over-the-counter for fever and pain. Its overdosing causes oxidative stress and subsequent acute liver damage. In the present study, we scrutinized the protective effect of metformin co-treatment in APAP induced blood and liver sub-acute toxicity. This is a pre-clinical study in which male Wistar Rats (BW: 300 ± 20 g) were orally co-treated with APAP (1 g/kg/day) and metformin (300 mg/kg/day) for 28-days. Pro- and anti-oxidant markers viz reactive oxygen species, protein carbonyl, malondialdehyde (MDA), the ferric reducing ability of plasma (FRAP), plasma membrane redox system(PMRS) and reduced glutathione (GSH) were evaluated in blood. Additionally, in liver tissue, catalase (CAT), superoxide dismutase (SOD), MDA and GST level were also evaluated. Histological study and estimation of alanine aminotransferase (ALT), and aspartate aminotransferase (AST) level in serum were performed. APAP induces pro-oxidant markers as well as reduces anti-oxidant markers in blood and liver. Hepatic tissues degeneration and vacuolization of hepatocytes were evident after APAP treatment. Metformin treatment reduces pro-oxidant markers as well as increases anti-oxidant markers in both tissues. It also improves liver tissue architecture after treatment. The outcome of this study suggests that metformin has protective capability against APAP-induced blood and liver toxicity. Thus, metformin co-treatment with APAP attenuates oxidative stress and its consequences.

Chronic Embryo-Larval Exposure of Fathead Minnows to the Pharmaceutical Drug Metformin: Survival, Growth, and Microbiome Responses.

Metformin is a glucose-lowering drug commonly found in municipal wastewater effluents (MWWEs). The present study investigated the chronic effects of metformin in early-life stages of the fathead minnow (Pimephales promelas). Endpoints assessed were growth, survival, and deformities. The larval gut microbiome was also examined using 16 S ribosomal RNA gene amplicon sequencing to determine microbial community composition and alpha and beta diversity. Eggs and larvae were exposed to metformin measured concentrations (mean [standard deviation]) of 0.020 (0.017) µg/L (for controls) and 3.44 (0.23), 33.6 (1.6), and 269 (11) µg/L in a daily static-renewal setup, with 20 embryos per beaker. The low and middle metformin exposure concentrations represent river and MWWE concentrations of metformin. To detect small changes in growth, we used 18 replicate beakers for controls and 9 replicates for each metformin treatment. Over the 21-d exposure (5 d as embryos and 16 d posthatch [dph]), metformin did not affect survival or growth of larval fish. Hatch success, time to hatch, deformities in hatched fry, and survival were similar across all treatments. Growth (wet wt, length, and condition factor) assessed at 9 and 16 dph was also unaffected by metformin. Assessment of the microbiome showed that the larvae microbiome was dominant in Proteobacteria and Firmicutes, with small increases in Proteobacteria and decreases in Firmicutes with increasing exposure to metformin. No treatment effects were found for microbiome diversity measures. Control fish euthanized with the anesthetic tricaine methane sulfonate had decreased alpha diversity compared to those sampled by spinal severance. This experiment demonstrates that metformin at environmentally relevant concentrations (3.44 and 33.6 µg/L) and at 10 times MWWE concentrations (269 µg/L) does not adversely affect larval growth or gut microbiome in this ubiquitous freshwater fish species. Environ Toxicol Chem 2022;41:635-647. © 2021 Her Majesty the Queen in Right of Canada. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC. Reproduced with the permission of the Minister of Environment and Climate Change Canada.

Palliative effects of metformin on testicular damage induced by triptolide in male rats.

As a widely existing traditional Chinese medicine component, TP (triptolide) has serious reproductive toxicity which causes severe damage to the reproductive system and limits its application prospect. TP and MET (metformin) have shown great potential in combined with each other in anticancer and anti-inflammatory. Whether metformin can resist the reproductive toxicity caused by triptolide, the effects of MET on TP-induced reproductive capacity has not been reported. In this study, metformin was used to investigate the therapeutic effect on reproductive toxicity induced by TP in rat. The results showed that metformin had significant therapeutic effects on oxidative stress damage, destruction of the blood-testosterone barrier and apoptosis. And it proved that its therapeutic effect is mainly to restore the structural and functional stability of testis through antioxidant stress. It will provide guidance for the treatment of reproductive toxicity caused by TP and the adjuvant detoxification of TP application.

Antidiabetic drug metformin disrupts the embryogenesis in zebrafish through an oxidative stress mechanism.

In recent years, the consumption of metformin has increased not only due to the higher prevalence of type 2 diabetes, but also due to their usage for other indications such as cancer and polycystic ovary syndrome. Consequently, metformin is currently among the highest drug by weight released into the aquatic environments. Since the toxic effects of this drug on aquatic species has been scarcely explored, the aim of this work was to investigate the influence of metformin on the development and redox balance of zebrafish (Danio rerio) embryos. For this purpose, zebrafish embryos (4 hpf) were exposed to 1, 10, 20, 30, 40, 50, 75 and 100 µg/L metformin until 96 hpf. Metformin significantly accelerated the hatching process in all exposure groups. Moreover, this drug induced several morphological alterations on the embryos, affecting their integrity and consequently leading to their death. The most frequent malformations found on the embryos included malformation of tail, scoliosis, pericardial edema and yolk deformation. Regarding oxidative balance, metformin significantly induced the activity of antioxidant enzymes and the levels of oxidative damage biomarkers. However, our IBR analisis demonstrated that oxidative damage biomarkers got more influence over the embryos. Together these results demonstrated that metformin may affect the embryonic development of zebrafish and that oxidative stress may be involved in the generation of this embryotoxic process.

Developmental phenotypic and transcriptomic effects of exposure to nanomolar levels of metformin in zebrafish.

Metformin is found in the majority of lakes and streams in the United States, leading to widespread environmental exposure. Results of the present study indicate that extended duration metformin exposure at critical developmental periods leads to decreased survival rates in zebrafish (danio rerio), an NIH approved human model. Significant abnormalities are seen with extended duration metformin exposure from 4 h post fertilization up to 5 days post fertilization, although short term metformin exposure for 24 h at 4-5 days post fertilization did not lead to any significant abnormalities. Both extended and short term duration did however have an impact on locomotor activity of zebrafish, and several genes involved in neurological and cardiovascular development were differentially expressed after exposure to metformin. The changes seen in behavior, gene expression and morphological abnormalities caused by metformin exposure should be examined further in future studies in order to assess their potential human health implications as metformin prescriptions continue to increase worldwide.