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.

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.

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.

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.

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.

Metformin impairs homing ability and efficacy of mesenchymal stem cells for cardiac repair in streptozotocin-induced diabetic cardiomyopathy in rats.

Bone marrow-derived mesenchymal stem cells (BM-MSCs) have demonstrated potential in treating diabetic cardiomyopathy. However, patients with diabetes are on multiple drugs and there is a lack of understanding of how transplanted stem cells would respond in presence of such drugs. Metformin is an AMP kinase (AMPK) activator, the widest used antidiabetic drug. In this study, we investigated the effect of metformin on the efficacy of stem cell therapy in a diabetic cardiomyopathy animal model using streptozotocin (STZ) in male Wistar rats. To comprehend the effect of metformin on the efficacy of BM-MSCs, we transplanted BM-MSCs (1 million cells/rat) with or without metformin. Our data demonstrate that transplantation of BM-MSCs prevented cardiac fibrosis and promoted angiogenesis in diabetic hearts. However, metformin supplementation downregulated BM-MSC-mediated cardioprotection. Interestingly, both BM-MSCs and metformin treatment individually improved cardiac function with no synergistic effect of metformin supplementation along with BM-MSCs. Investigating the mechanisms of loss of efficacy of BM-MSCs in the presence of metformin, we found that metformin treatment impairs homing of implanted BM-MSCs in the heart and leads to poor survival of transplanted cells. Furthermore, our data demonstrate that metformin-mediated activation of AMPK is responsible for poor homing and survival of BM-MSCs in the diabetic heart. Hence, the current study confirms that a conflict arises between metformin and BM-MSCs for treating diabetic cardiomyopathy. Approximately 10% of the world population is diabetic to which metformin is prescribed very commonly. Hence, future cell replacement therapies in combination with AMPK inhibitors may be more effective for patients with diabetes.NEW & NOTEWORTHY Metformin treatment reduces the efficacy of mesenchymal stem cell therapy for cardiac repair during diabetic cardiomyopathy. Stem cell therapy in diabetics may be more effective in combination with AMPK inhibitors.

Metformin-Induced Epigenetic Toxicity in Zebrafish: Experimental and Molecular Dynamics Simulation Studies.

The increased detection of many prescription drugs in aquatic environments has heightened concerns of their potential ecotoxicological effects. In this study, the effects of metformin (MEF) exposure on tissue accumulation, gene expression, and global DNA methylation (GDM) in zebrafish were investigated. The toxic mechanism of MEF exposure was simulated by molecular dynamics (MD) to reveal any conformational changes to DNA methyltransferase 1 (DNMT1). The results showed MEF accumulation in the gills, gut, and liver of zebrafish after 30 days of exposure, and the bioaccumulation capacity was in the order of gut > liver > gills. After a 30 day recovery period, MEF could still be detected in zebrafish tissues in groups exposed to MEF concentrations ≥ 10 µg/L. Moreover, the liver was the main site of GDM, and the restoration of GDM in the liver was slower than that in the gut and gills during the recovery period. Furthermore, MEF could induce the abnormal expression of CYP3A65, GSTM1, p53, and DNMT1 genes in the liver due to the formation of hydrogen bonds between MEF and the protein residues of those genes. The MD simulation allowed for the mechanistic determination of MEF-induced three-dimensional (3D) conformational changes and changes to the catalytic activity of DNMT1.

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.

Diazepam, metformin, omeprazole and simvastatin: a full discussion of individual and mixture acute toxicity.

High consumption of drugs, combined with their presence in the environment, raises concerns about its consequences. Even though researches are often engaged in analyzing substances separately, that is not the environmental reality. Therefore, the aim of this study was to investigate the acute toxicity of the pharmaceuticals simvastatin, metformin, omeprazole and diazepam, and all possible mixtures between them, to the organism Aliivibrio fischeri, verifying possible synergistic or antagonistic effects and assessing byproducts formation. In terms of individual toxicity, omeprazole is the most toxic of the active ingredients, followed by simvastatin, diazepam and, finally, metformin. When the toxicity of mixtures was tested, synergism, antagonism and hormesis were perceived, most probably generated due to byproducts formation. Moreover, it was observed that even when compounds are at concentrations below the non-observed effect concentration (NOEC), there may be toxicity to the mixture. Hence, this work points to the urgent need for more studies involving mixtures, since chemicals are subject to interactions and modifications, can mix, and potentiate or nullify the toxic effect of each other.

Energy metabolism modulation by biguanides in comparison with rotenone in rat liver and heart.

The biguanide metformin, a widely used antidiabetic drug, has received great interest in oncology research in recent years after an epidemiological study showed a link between metformin treatment and a reduced cancer risk in diabetic patients. Since mitochondrial metabolism has become a target for possible cancer therapeutic approaches, especially for tumors relying on oxidative metabolism, mitochondrial complex I inhibition is under discussion to be responsible for the anti-cancer effect of metformin. Rotenone, a well-known strong mitochondrial complex I inhibitor, yet associated with toxic effects, has also shown anti-cancer activity. Thus, we compared metformin and phenformin, another biguanide previously on the market as antidiabetic, with rotenone, to elucidate potential mechanisms rendering biguanides apparently less toxic than rotenone. Therefore, we conducted in vivo rat studies with metformin and phenformin, based on an experimental design previously described for mechanistic investigations of the effects of rotenone, including blood and tissue analysis, histopathology and gene expression profiling. These investigations show that the mechanistic profile of phenformin appears similar to that of rotenone, yet at a quantitatively reduced level, whereas metformin displays only transient similarities after one day of treatment. A potential reason may be that metformin, but not rotenone or phenformin, self-limits its entry into mitochondria due to its molecular properties. Thus, our detailed molecular characterization of these compounds suggests that inhibition of mitochondrial functions can serve as target for an anti-cancer mode of action, but should be self-limited or balanced to some extent to avoid exhaustion of all energy stores.

Concentration-dependent metabolic effects of metformin in healthy and Fanconi anemia lymphoblast cells.

Metformin (MET) is the drug of choice for patients with type 2 diabetes and has been proposed for use in cancer therapy and for treating other metabolic diseases. More than 14,000 studies have been published addressing the cellular mechanisms affected by MET. However, several in vitro studies have used concentrations of the drug 10-100-fold higher than the plasmatic concentration measured in patients. Here, we evaluated the biochemical, metabolic, and morphologic effects of various concentrations of MET. Moreover, we tested the effect of MET on Fanconi Anemia (FA) cells, a DNA repair genetic disease with defects in energetic and glucose metabolism, as well as on human promyelocytic leukemia (HL60) cell lines. We found that the response of wild-type cells to MET is concentration dependent. Low concentrations (15 and 150 µM) increase both oxidative phosphorylation and the oxidative stress response, acting on the AMPK/Sirt1 pathway, while the high concentration (1.5 mM) inhibits the respiratory chain, alters cell morphology, becoming toxic to the cells. In FA cells, MET was unable to correct the energetic/respiratory defect and did not improve the response to oxidative stress and DNA damage. By contrast, HL60 cells appear sensitive also at 150 µM. Our findings underline the importance of the MET concentration in evaluating the effect of this drug on cell metabolism and demonstrate that data obtained from in vitro experiments, that have used high concentrations of MET, cannot be readily translated into improving our understanding of the cellular effects of metformin when used in the clinical setting.

Clinical and Laboratory Features of Metformin Intoxication in Suicidal Patients Attending an Intensive Care Unit.

INTRODUCTION: This study has been performed for the purpose of researching the complications occurred at patients who took metformin overdose in an attempt to suicide. None of the patients has the diagnosis of diabetes mellitus and never used metformin. MATERIALS AND METHODS: This retrospective cross-sectional study was carried out with 21 patients who has neither diagnosed diabetes mellitus nor taken metformin for suicide before. RESULTS: It was observed that there is a moderate, negative (r = -0.63) statistically significant correlation (P < 0.001) between the time of applying to the hospital and arterial blood pH at the arrival and a statistically significant positive mild correlation (P < 0.041) between applying and blood lactate level (r = 0.45), and a moderate positive (r = 0.63) and statistically significant correlation (P < 0.001) between the total metformin dose and blood lactate level at the arrival and a positive, moderate (r = 0.68) significant correlation (P < 0.001) between the creatinine and metformin dose at the arrival. Lactic acidosis has been detected at 8 of 21 patients, 6 patients were hemodialized, 2 patients needed mechanical ventilation, and 2 patients died. It is observed that there is no mortality for early hemodialized patients. CONCLUSION: The most important reason of the mortality in patients who has metformin intoxication is metformin-associated lactic acidosis (MALA). It was considered that hemodialysis therapy could be effective in MALA.

Combination of Solamargine and Metformin Strengthens IGFBP1 Gene Expression Through Inactivation of Stat3 and Reciprocal Interaction Between FOXO3a and SP1.

BACKGROUND/AIMS: Solamargine, one natural photochemical component from traditional plants, has been shown to have anti-cancers properties. We previously showed that solamargine inhibited the growth of non-small-cell lung cancer (NSCLC) cells through suppression of prostaglandin E2 (PGE2) receptor EP4 gene and regulation of downstream signaling pathways. However, the detailed mechanism underlying this, especially in combination of metformin, a known AMPK activator, still remained to be determined. METHODS: Cell viability was measured using a 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) and colorimetric 5-bromo-2-deoxyuridine (BrdU) ELISA methods, respectively. Western blot analysis and immunohistochemistry were performed to examine the phosphorylation and protein expressions of signal transducer and activator of transcription 3 (Stat3), SP1, forkhead box O3a (FOXO3a), and insulin-like growth factor (IGF)-IGF binding protein 1 (IGFBP1). The expression of IGFBP1 mRNA was measured by quantitative real time PCR (qRT-PCR). Silencing of FOXO3a and IGFBP1 were examined by siRNA procedures. Exogenously expression of SP1, FOXO3a, and IGFBP1 were carried out by transient transfection assays. The promoter activity of IGFBP1 was tested using Secrete-PairTM Dual Luminescence Assay Kit. A xenografted tumor model was used to further test the effect of solamargine in combining with metformin in vivo. RESULTS: We further demonstrated that solamargine inhibited growth and induced cell cycle arrest in other NSCLC cell lines. Through mechanism-based approaches, we showed that solamargine decreased the phosphorylation of Stat3; In addition, solamargine induced FOXO3a, whereas reduced SP1 protein levels; all of which were abrogated in cells with overexpressed Stat3 gene. Interestingly, there is interaction between FOXO3a and SP1. Moreover, solamargine increased mRNA, protein expression and promoter activity of IGFBP1, which was not observed in cells with overexpressed SP1 or with silenced FOXO3a genes. Finally, ablation of IGFBP1 expression by siRNA blocked the effect of solamargine on cell growth inhibition. More importantly, there was a synergy of combination of solamargine and metformin. Similar findings were also observed in vivo. CONCLUSION: Our results show that solamargine increases IGFBP1 gene expression through inactivation of Stat3, followed by regulation and reciprocal interaction of FOXO3a and SP1 in vitro and in vivo. This ultimately leads to suppression of human lung cancer cell growth. Moreover, this is a synergy of solamargine in combination with metformin in this process. This study unravels a novel mechanism underlying the anti-lung cancer effects of solamargine in combination of metformin, and suggests a potential new lung cancer associated therapy.

Effect of metformin and celecoxib on cytotoxicity and release of GDF-15 from human mesenchymal stem cells in high glucose condition.

Mesenchymal stem cells (MSCs) have therapeutic potential for treatment of diabetes. However, in vitro behavior of MSCs in high glucose condition as well as presence of glucose lowering agents is not fully understood. Because MSCs have an important role in tissue repair, we examined the effects of metformin and celecoxib on viability of MSCs in different glucose conditions. MSCs, from umbilical cord blood, were cultured in normoglycemic (glucose 5.5 mM), midglycemic (glucose 10 mM), and hyperglycemic (glucose 25 mM) conditions, and the cell viability was evaluated by MTT assay. The cytotoxicity and secretion of GDF-15 were further tested in MSCs treated with metformin and celecoxib in various glucose concentrations. Our results showed that high glucose condition lowered viability of MSCs. Metformin treatment also inhibited proliferation of MSCs, but its toxicity was not changed in high glucose condition. Celecoxib induced cytotoxicity in MSCs, and the toxicity was increased in high glucose condition. Metformin and celecoxib induced release from MSCs; however, high glucose inhibited the metformin-induced GDF-15 release. These findings suggested that metformin did not increase the cytotoxicity of high glucose condition in MSCs. Moreover, celecoxib treatment in diabetic condition can reduce the viability of MSCs to proliferate and regenerate perhaps via change in release of GDF-15.

ROS Production and ERK Activity Are Involved in the Effects of d-ß-Hydroxybutyrate and Metformin in a Glucose Deficient Condition.

Hypoglycemia, a complication of insulin or sulfonylurea therapy in diabetic patients, leads to brain damage. Furthermore, glucose replenishment following hypoglycemic coma induces neuronal cell death. In this study, we investigated the molecular mechanism underlying glucose deficiency-induced cytotoxicity and the protective effect of d-ß-hydroxybutyrate (D-BHB) using SH-SY5Y cells. The cytotoxic mechanism of metformin under glucose deficiency was also examined. Cell viability under 1 mM glucose (glucose deficiency) was significantly decreased which was accompanied by increased production of reactive oxygen species (ROS) and decreased phosphorylation of extracellular signal-regulated kinase (ERK) and glycogen synthase 3 (GSK3ß). ROS inhibitor reversed the glucose deficiency-induced cytotoxicity and restored the reduced phosphorylation of ERK and GSK3ß. While metformin did not alter cell viability in normal glucose media, it further increased cell death and ROS production under glucose deficiency. However, D-BHB reversed cytotoxicity, ROS production, and the decrease in phosphorylation of ERK and GSK3ß induced by the glucose deficiency. ERK inhibitor reversed the D-BHB-induced increase in cell viability under glucose deficiency, whereas GSK3ß inhibitor did not restore glucose deficiency-induced cytotoxicity. Finally, the protective effect of D-BHB against glucose deficiency was confirmed in primary neuronal cells. We demonstrate that glucose deficiency-induced cytotoxicity is mediated by ERK inhibition through ROS production, which is attenuated by D-BHB and intensified by metformin.

Metformin, besides exhibiting strong in vivo anti-inflammatory properties, increases mptp-induced damage to the nigrostriatal dopaminergic system.

Metformin is a widely used oral antidiabetic drug with known anti-inflammatory properties due to its action on AMPK protein. This drug has shown a protective effect on various tissues, including cortical neurons. The aim of this study was to determine the effect of metformin on the dopaminergic neurons of the substantia nigra of mice using the animal model of Parkinson's disease based on the injection of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, an inhibitor of the mitochondrial complex I. In vivo and in vitro experiments were used to study the activation of microglia and the damage of the dopaminergic neurons. Our results show that metformin reduced microglial activation measured both at cellular and molecular levels. Rather than protecting, metformin exacerbated dopaminergic damage in response to MPTP. Our data suggest that, contrary to other brain structures, metformin treatment could be deleterious for the dopaminergic system. Hence, metformin treatment may be considered as a risk factor for the development of Parkinson's disease.

Extracorporeal Treatment for Metformin Poisoning: Systematic Review and Recommendations From the Extracorporeal Treatments in Poisoning Workgroup.

BACKGROUND: Metformin toxicity, a challenging clinical entity, is associated with a mortality of 30%. The role of extracorporeal treatments such as hemodialysis is poorly defined at present. Here, the Extracorporeal Treatments In Poisoning workgroup, comprising international experts representing diverse professions, presents its systematic review and clinical recommendations for extracorporeal treatment in metformin poisoning. METHODS: A systematic literature search was performed, data extracted, findings summarized, and structured voting statements developed. A two-round modified Delphi method was used to achieve consensus on voting statements and RAND/UCLA Appropriateness Method to quantify disagreement. Anonymized votes and opinions were compiled and discussed. A second vote determined the final recommendations. RESULTS: One hundred seventy-five articles were identified, including 63 deaths: one observational study, 160 case reports or series, 11 studies of descriptive cohorts, and three pharmacokinetic studies in end-stage renal disease, yielding a very low quality of evidence for all recommendations. The workgroup concluded that metformin is moderately dialyzable (level of evidence C) and made the following recommendations: extracorporeal treatment is recommended in severe metformin poisoning (1D). Indications for extracorporeal treatment include lactate concentration greater than 20 mmol/L (1D), pH less than or equal to 7.0 (1D), shock (1D), failure of standard supportive measures (1D), and decreased level of consciousness (2D). Extracorporeal treatment should be continued until the lactate concentration is less than 3 mmol/L (1D) and pH greater than 7.35 (1D), at which time close monitoring is warranted to determine the need for additional courses of extracorporeal treatment. Intermittent hemodialysis is preferred initially (1D), but continuous renal replacement therapies may be considered if hemodialysis is unavailable (2D). Repeat extracorporeal treatment sessions may use hemodialysis (1D) or continuous renal replacement therapy (1D). CONCLUSION: Metformin poisoning with lactic acidosis appears to be amenable to extracorporeal treatments. Despite clinical evidence comprised mostly of case reports and suboptimal toxicokinetic data, the workgroup recommended extracorporeal removal in the case of severe metformin poisoning.

Human mesenchymal stem cells as a novel platform for simultaneous evaluation of cytotoxicity and genotoxicity of pharmaceuticals.

The in vitro micronucleus test is a well-known test for the screening of genotoxic compounds. However until now, most studies have been performed on either human peripheral lymphocytes or established cancer cell lines. This study provides human mesenchymal stem cells as an alternative to the conventional micronucleus test. We grew umbilical cord mesenchymal stem cells (UC-MSCs) on coverslips eliminating the cumbersome technique involving hypotonic treatment, fixation and preparing smears required for suspension culture (lymphocytes). The background frequency of nuclear blebs and micronuclei in UC-MSCs was found to be 7±5, in lymphocytes 16±3.5 and 9±3 and that for A549 cell line was 65±5 and 15±5 per 1000 cells, respectively, suggesting differences in the repair mechanism of normal and cancer cell lines. We inspected the cytotoxic and genotoxic effects of two known mutagens, mitomycin-C and hydrogen peroxide (H2O2), on UC-MSCs, lymphocytes and A549 cells. Treatment with mitomycin-C and H2O2 demonstrated drastic differences in the degree of cytotoxicity and genotoxicity suggesting a constitutional difference between normal and cancer cells. In addition we tested two solvents, dimethyl sulfoxide (DMSO) and ethanol, and two drugs, metformin and rapamycin. DMSO above 1% was found to be cytotoxic and genotoxic, whereas ethanol at same concentration was neither cytotoxic nor genotoxic indicating the minimal non-toxic level of the solvents. This study thus offers UC-MSCs as a better substitute to peripheral lymphocytes and cancer cell lines for high throughput screening of compounds and reducing the animal studies.

Intracerebroventricular metformin decreases body weight but has pro-oxidant effects and decreases survival.

Metformin (Met), which is an insulin-sensitizer, decreases insulin resistance and fasting insulin levels. The precise molecular target of Met is unknown; however, several reports have shown an inhibitory effect on mitochondrial complex I of the electron transport chain (ETC), which is a related site for reactive oxygen species production. In addition to peripheral effects, Met is capable of crossing the blood-brain barrier, thus regulating the central mechanism involved in appetite control. The present study explores the effects of intracerebroventricular (i.c.v.) infusion of Met on ROS production on brain, insulin sensitivity and metabolic and oxidative stress outcomes in CF1 mice. Metformin (Met 50 and 100 µg) was injected i.c.v. in mice daily for 7 days; the brain mitochondrial H2O2 production, food intake, body weight and fat pads were evaluated. The basal production of H2O2 of isolated mitochondria from the hippocampus and hypothalamus was significantly increased by Met (100 µg). There was increased peripheral sensitivity to insulin (Met 100 µg) and glucose tolerance tests (Met 50 and 100 µg). Moreover, Met decreased food intake, body weight, body temperature, fat pads and survival rates. Additionally, Met (1, 4 or 10 mM) decreased mitochondrial viability and increased the production of H2O2 in neuronal cell cultures. In summary, our data indicate that a high dose of Met injected directly into the brain has remarkable neurotoxic effects, as evidenced by hypothermia, hypoglycemia, disrupted mitochondrial ETC flux and decreased survival rate.