Detailed cytotoxicity assessment of the formulated herbicide roundup classic and its constituents

Cytotoxicity of the globally market-leading herbicide ROUNDUP CLASSIC formulation and its components such as the active ingredient glyphosate and the formulating agent POEA (a mixture of polyethoxylated tallow amines) were investigated on the murine neuroectodermal stem cell-like (NE-4C) and osteoblastic (MC3T3-E1) cell lines. The cytotoxic and genotoxic effects on cell viability and cell cycles were evaluated based on the results of flow cytometry, enzymatic-assays, and alkaline single cell gel electrophoresis (Comet) assays, furthermore, the effects on cell morphology and dynamic mass redistribution of cellular contents were assessed with the use of the label-free Epic BenchTop optical biosensor on MC3T3-E1 cells adhered on the surface of the biosensor. Differences in the sensitivity of the investigated cell lines were detected, while the MC3T3-E1 cell line indicated less sensitivity to the effects of the treatments. Furthermore, differences were also observed in the sensitivity of the performed assays. The order of the inhibitory potency of the investigated compounds was as follows: glyphosate IPA salt << ROUNDUP CLASSIC < POEA. The applied Epic technique provides an effective tool for the real-time detection of cytotoxicity

A newly identified specific biological activity of glyphosate - inhibition of RGD-binding integrins

In this study we investigated the inhibitory effect of the widely used broad-spectrum herbicide active ingredient glyphosate and its related analogues on αVβ3 integrin binding to the shortest oligopeptide recognizing motif of integrins, the arginine-glycine-aspartic acid (RGD) sequence. Integrin binding characteristics were assessed in a modified enzyme linked immunosorbent assay (ELISA) and by a label-free optical biosensor technique. At 22 mM, glyphosate reached full inhibition of αVβ3, and the inhibitory activity of its main metabolite, aminomethylphosphonic acid (AMPA) was also above 95%, while another environmentally relevant metabolite, sarcosine exerted only a weaker effect, approximately 35% inhibition. In turn, the half maximal inhibitory concentration (IC50) of glyphosate and AMPA were reported to be 2.7±0.5 mM and 1.3±0.2 mM, respectively. The inhibitory effects of the other related compounds investigated (acetylglycine, glycine and iminodiacetic acid) at the same concentration, 22 mM were below 50%. Inhibitory effects on cell adhesion to RGD-modified surfaces by whole cells containing several types of RGD-binding integrins including αVβ3 were detected using the biosensor technique, where the integrin antagonist activity of glyphosate was also demonstrated

Comparative Assessment of the Inhibitory Potential of the Herbicide Glyphosate and Its Structural Analogs on RGD-Specific Integrins Using Enzyme-Linked Immunosorbent Assays

Transmembrane glycoprotein integrins play crucial roles in biochemical processes, and by their inhibition or activation, different signal pathways can be disrupted, leading to abnormal physiological functions. We have previously demonstrated the inhibitory effect of glyphosate herbicide’s active ingredient on cell adhesion and its αvβ3 integrin antagonist effect. Therefore, it appeared particularly exciting to investigate inhibition of glyphosate and its metabolites on a wider range of Arg-Gly-Asp (RGD) binding integrins, namely αvβ3, α5β1 and αllbβ3. Thus, the purpose of this study was to assess how extended the inhibitory effect observed for glyphosate on the integrin αvβ3 is in terms of other RGD integrins and other structurally or metabolically related derivatives of glyphosate. Five different experimental setups using enzyme-linked immunosorbent assays were applied: (i) αvβ3 binding to a synthetic polymer containing RGD; (ii) αvβ3 binding to its extracellular matrix (ECM) protein, vitronectin; (iii) α5β1 binding to the above polymer containing RGD; (iv) αllbβ3 binding to its ECM protein, fibrinogen and (v) αvβ3 binding to the SARS-CoV-2 spike protein receptor binding domain. Total inhibition of αvβ3 binding to RGD was detected for glyphosate and its main metabolite, aminomethylphosphonic acid (AMPA), as well as for acetylglycine on α5β1 binding to RGD

Cytotoxic effects of Roundup Classic and its components on NE-4C and MC3T3-E1 cell lines determined by biochemical and flow cytometric assays

Cytotoxic effects of the market leading broad-spectrum, synthetic herbicide product Roundup Classic, its active ingredient glyphosate (in a form of its isopropylamine (IPA) salt) and its formulating surfactant polyethoxylated tallowamine (POE-15) were determined on two murine cell lines, a neuroectodermal stem cell-like (NE-4C) and a high alkaline phosphatase activity osteoblastic cell line (MC3T3-E1). Cytotoxicity, genotoxicity, effects on cell viability and cell cycles were examined in five flow cytometry tests, the two former of which were compared by the enzymatic-assay and the alkaline single cell gel electrophoresis (Comet) assay. All of the tests indicated the NE-4C cells being more sensitive, than the MC3T3-E1 cell line to the treatments with the target compounds. Higher sensitivity differences were detected in the viability test by flow cytometry (7–9-fold), than by the MTT assay (1.5–3-fold); in the genotoxicity test by the Comet assay (3.5–403-fold), than by the DNA-damage test (9.3–158-fold); and in the apoptosis test by the Annexin V dead cell kit (1.1–12.7-fold), than by the Caspase 3/7 kit (1–6.5-fold). Cell cycle assays indicated high count of cells (~70%) in the G0/G1 phase for MC3T3-E1 cells, than in NE-4C cell (~40%) after 24 h. The order of the inhibitory potency of the target substances has unequivocally been POE-15 > Roundup Classic > > glyphosate IPA salt

Epigallocatechin-gallate tailors the cell adhesivity of fibronectin coatings in oxidation and concentration-dependent manner

Fibronectin is an extracellular matrix component that plays a significant role in many physiological processes, such as cell adhesion, growth, differentiation, and migration. In this study, we revealed the interaction between this important protein and the widely studied natural active substance green tea polyphenol epigallocatechin-gallate (EGCG) and its oxidized form. Furthermore, we investigated the kinetics of cancer cell adhesion on the polyphenol-treated fibronectin coatings. We applied a high- throughput, label-free optical biosensor capable of monitoring the above processes in real time with an excellent signal-to-noise ratio. Our results show that EGCG and its oxidized form bind to fibronectin in a concentration-dependent manner and can form multilayers as well. Furthermore, both polyphenol forms inhibited cellular adhesion, however, the effect was more pronounced in the case of the oxidized form. The results were compared to the measurements performed on bare biosensor surfaces without fibronectin, and the roles of oxidation were investigated. It is suggested that the polyphenols can interact and block important cell adhesion protein motifs and affect the rigidity of the layers as well. Moreover, a novel molecular scale active mechanism involving the disulfide bridges of fibronectin was proposed to explain the recorded kinetic signals and highlight that these proteins can be active participants in the molecular scale transformations affecting adhesion

Population distributions of single-cell adhesion parameters during the cell cycle from high-throughput robotic fluidic force microscopy

Single-cell adhesion plays an essential role in biological and biomedical sciences, but its precise measurement for a large number of cells is still a challenging task. At present, typical force measuring techniques usually offer low throughput, a few cells per day, and therefore are unable to uncover phenomena emerging at the population level. In this work, robotic fluidic force microscopy (FluidFM) was utilized to measure the adhesion parameters of cells in a high-throughput manner to study their population distributions in-depth. The investigated cell type was the genetically engineered HeLa Fucci construct with cell cycle-dependent expression of fluorescent proteins. This feature, combined with the high-throughput measurement made it possible for the first time to characterize the single-cell adhesion distributions at various stages of the cell cycle. It was found that parameters such as single-cell adhesion force and energy follow a lognormal population distribution. Therefore, conclusions based on adhesion data of a low number of cells or treating the population as normally distributed can be misleading. Moreover, we found that the cell area was significantly the smallest, and the area normalized maximal adhesion force was significantly the largest for the colorless cells (the mitotic (M) and early G1 phases). Notably, the parameter characterizing the elongation of the cells until the maximum level of force between the cell and its substratum was also dependent on the cell cycle, which quantity was the smallest for the colorless cells. A novel parameter, named the spring coefficient of the cell, was introduced as the fraction of maximal adhesion force and maximal cell elongation during the mechanical detachment, which was found to be significantly the largest for the colorless cells. Cells in the M phase adhere in atypical way, with so-called reticular adhesions, which are different from canonical focal adhesions. We first revealed that reticular adhesion can exert a higher force per unit area than canonical focal adhesions, and cells in this phase are significantly stiffer. The possible biological consequences of these findings were also discussed, together with the practical relevance of the observed population-level adhesion phenomena