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

Jelölésmentes optikai bioszenzorok a sejtadhézió vizsgálatában

A sejtadhézió kulcsfontosságú biológiai jelenség, melynek során egy sejt specifikus receptor-ligandum kapcsolatokon keresztül önmagát egy megfelelő aljzatra vagy másik sejtre kihorgonyozva, sejtvázát aktívan átszervezve kiterül, és felveszi a sejttípusra jellemző morfológiát. Fundamentális jelentőségénél fogva a sejtadhézió minél részletesebb leírása és megértése számos interdiszciplináris tudományág, így például a biofizika, gyógyszertan, vagy az orvos- és anyagtudomány egyik alapvető célja és érdeke. Mindennek ellenére a legtöbb, a területen alkalmazott vizsgálati technika az eredendően dinamikus természetű adhéziót egyetlen időpillanatban vagy rossz időbeli felbontással, tehát hiányosan és szegényesen képes csak karakterizálni. Kivételt képeznek ez alól a jelölésmentes bioszenzorok, melyek felületközeli folyamatok kinetikájának kvázi perturbációmentes követését teszik lehetővé. A sejtadhézió vizsgálatára különösen alkalmasnak bizonyultak az evaneszcens tér-alapú optikai bioszenzorok, mivel i) kizárólag a folyamat szempontjából legrelevánsabb, a tapadási felületként szolgáló sík szenzorhoz legközelebbi ~150 nm-es tartományban érzékenyek, illetve ii) jelük egy integrált jel, mely nem csak az átlagos kontaktterülettől, hanem az azon belüli optikai sűrűségtől is függ, így az adhézió mértékének kiváló mérőszámaként használható. Az újszerű Epic BenchTop nagy áteresztőképességű optikai bioszenzort [1] az elsők között használtuk a sejtahézió tanulmányozására. Első ízben HeLa sejtek adhéziós kinetikájának az adhéziós ligandok felületi sűrűségétől való függését vizsgáltuk. A kiváló minőségű adatokat kinetikai elemzésnek alávetve megállapítottuk, hogy a folyamat sebességi állandója nem függ a ligandsűrűségtől, az adhézió mértéke viszont igen. Az adhéziós receptorok és a ligandum közötti kölcsönhatást egyszerű monovalens kötési folyamatként modelleztük, és az utóbbi adatokat megillesztve meghatároztuk a receptor-ligandum kölcsönhatás disszociációs állandóját. Ez 30 μM-nak adódott, melyet irodalmi adatokkal összevetve reális értéknek találtunk [2]. Bár számos esetben a sejtadhézió kinetikája leírható egy szimmetrikus szigmoiddal, egyre több eredményünk mutat abba az irányba, hogy felületkémiai módosítások vagy egyes kis molekulás hatóanyagokkal történő kezelések hatására a sejtadhézió nem triviális, esetenként pedig még csak nem is monoton kinetikát követ. Mindez tovább hangsúlyozza, hogy a kinetikai adatrögíztés mindenféle sejtes kísérletben alapvető fontosságú lehet

Environmental and Toxicological Impacts of Glyphosate with Its Formulating Adjuvant

Environmental and toxicological characteristics of formulated pesticides may substantially differ from those of their active ingredients or other components alone. This phenomenon is demonstrated in the case of the herbicide active ingredient glyphosate. Due to its extensive application, this active ingredient was found in surface and ground water samples collected in Békés Country, Hungary, in the concentration range of 0.54–0.98 ng/ml. The occurrence of glyphosate appeared to be somewhat higher at areas under intensive agriculture, industrial activities and public road services, but the compound was detected at areas under organic (ecological) farming or natural grasslands, indicating environmental mobility. Increased toxicity of the formulated herbicide product Roundup compared to that of glyphosate was observed on the indicator aquatic organism Daphnia magna Straus. Acute LC50 values of Roundup and its formulating adjuvant polyethoxylated tallowamine (POEA) exceeded 20 and 3.1 mg/ml, respectively, while that of glyphosate (as isopropyl salt) was found to be substantially lower (690-900 mg/ml) showing good agreement with literature data. Cytotoxicity of Roundup, POEA and glyphosate has been determined on the neuroectodermal cell line, NE-4C measured both by cell viability test and holographic microscopy. Acute toxicity (LC50) of Roundup, POEA and glyphosate on NE-4C cells was found to be 0.013±0.002%, 0.017±0.009% and 6.46±2.25%, respectively (in equivalents of diluted Roundup solution), corresponding to 0.022±0.003 and 53.1±18.5 mg/ml for POEA and glyphosate, respectively, indicating no statistical difference between Roundup and POEA and 2.5 orders of magnitude difference between these and glyphosate. The same order of cellular toxicity seen in average cell area has been indicated under quantitative cell visualization. The results indicate that toxicity of the formulated herbicide is caused by the formulating agent, but in some parameters toxicological synergy occurs between POEA and glyphosate

Simple and Automatic Monitoring of Cancer Cell Invasion into an Epithelial Monolayer Using Label-Free Holographic Microscopy

The invasiveness of cancer cells describes the metastasizing capability of a primary tumor. The straightforward detection and quantification of cancer cell invasion are important to predict the survival rate of a cancer patient and to test how anti-cancer compounds influence cancer progression. Digital holographic microscopy based M4 Holomonitor (HM) is a technique that allows the label-free monitoring of cell morphological and kinetical parameters in real-time. Here, a fully confluent epithelial monolayer derived from the African green monkey kidney (Vero) on a gelatin-coated surface was established, then HeLa cells were seeded on top of the monolayer, and their behavior was monitored for 24 h using HM. Several cancer cells showing invasiveness were detected during this period, while other HeLa cells did not show any signs of aggressivity. It was demonstrated that the invasion of single cancer cells is soundly observable and also quantifiable through monitoring parameters such as phase shift, optical volume, area, and motility, which parameters can easily be obtained and processed automatically. Based on the experimental data, the invasion speed of cancer cells entering the epithelial layer can be defined as the shrinking of detected single-cell volume per unit time. The invasion speed and its correlation with cell migration parameters were analyzed in depth. A clear linear relationship between migration and invasion speed was found, cancer cells with stronger migration have slower invasion speed. These results not only describe the effect of how cancer cells invade the underlying monolayer in contrast to non-invasive HeLa cells, but could help in future research to optimize drugs affecting cell invasibility in a fully automated, label-free and high-throughput manner