In vivo and in vitro tests for the detection of biochemical and ecotoxicological effects of the herbicide active ingredient glyphosate

Aquatic organisms are outstandingly exposed to water contaminants because of their unavoidable contact with xenobiotics, thus their exposure needs to be routinely monitored. Due to its extensive use, the herbicidal agrochemical active ingredient glyphosate realizes massive exposure, its toxic effects alone and in formulations were evaluated in different in vivo aquatic ecotoxicological tests on various algae species, freshwater biofilm communities, Daphnia magna, and Danio rerio, furthermore the possible cytotoxic, genotoxic, and hormonemodulating effects were evaluated in vitro on different cell lines and test organisms. Significant differences were detected in the individual and combined toxicity of glyphosate and its coformulants presented in the formulations, therefore various additives cannot be classified as unequivocally inactive components. The result of the in vivo testing proved higher toxicity for the formulating agent and the formulation compared to the individual effect of glyphosate, and significant differences in the sensitivity of test species, and the effects on the sexual development of fish were also observed. The performed in vitro assays on cell lines demonstrated the potential cytotoxic and genotoxic effects of glyphosate and its formulations, and some of the effects are the result of the individual toxicity of glyphosate

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

Determination of the mycotoxin zearalenone in water by immunofluorescence and total internal reflection ellipsometry methods

In the scope of project Aquafluosense developing prototypes of fluorescence-based instrumentation for in situ measurement of several characteristic parameters of water quality, an immunofluorescent method have been developed for the detection of several environmental xenobiotics, including mycotoxin zearalenone (ZON). ZON, produced by several plant pathogenic Fusarium species, has recently been identified as an emerging pollutant in surface water, presenting a hazard to aquatic ecosystems. Due to its physico-chemical properties, detection of ZON at low concentration in surface water is a challenging task. The 96-well microplate-based fluorescent instrument is capable to detect ZON in the concentration range of 0.4–400 ng mL-1 . The sensitivity and accuracy of the analytical methods has been demonstrated by comparative assessment with detection by total internal reflection ellipsometry

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

An Optical Planar Waveguide-Based Immunosensors for Determination of Fusarium Mycotoxin Zearalenone

A planar waveguide (PW) immunosensor working as a polarisation interferometer was developed for the detection of mycotoxin zearalenone (ZON). The main element of the sensor is an optical waveguide consisting of a thin silicon nitride layer between two thicker silicon dioxide layers. A combination of a narrow waveguiding core made by photolithography with an advanced optical set-up providing a coupling of circular polarised light into the PW via its slanted edge allowed the realization of a novel sensing principle by detection of the phase shift between the p- and s-components of polarised light propagating through the PW. As the p-component is sensitive to refractive index changes at the waveguide interface, molecular events between the sensor surface and the contacting sample solution can be detected. To detect ZON concentrations in the sample solution, ZON-specific antibodies were immobilised on the waveguide via an electrostatically deposited polyelectrolyte layer, and protein A was adsorbed on it. Refractive index changes on the surface due to the binding of ZON molecules to the anchored antibodies were detected in a concentration-dependent manner up to 1000 ng/mL of ZON, allowing a limit of detection of 0.01 ng/mL. Structurally unrelated mycotoxins such as aflatoxin B1 or ochratoxin A did not exert observable cross-reactivity

Enzyme-linked fluorescent immunoassay for monitoring the herbicide active ingredient glyphosate in water

Within a modular water quality assessment fluorimeter instrument family, a newly developed enzyme-linked fluorescent immunoassay has been utilized for the quantitative analytical measurement of the herbicide active ingredient glyphosate in surface water. The developed 96- well microplate-based competitive immunoassay with fluorescence detection provides a 2.5- fold lower limit of detection (LOD = 0.09 ng/mL) in the investigated concentration range of glyphosate (0–100 ng/mL) compared to the detection of visual absorbance signals. Additionally, fluorescence detection resulted in a wider dynamic range for glyphosate measurement. Matrix effect was not observed for the undiluted surface water samples, and cross-reaction was not detected between glyphosate and its main metabolite (Naminomethylphosphonic acid) and structurally similar compounds. The method allows rapid monitoring of glyphosate as a ubiquitous water contaminant of agricultural origin that can affect, due to its global use, both aquatic and terrestrial ecosystems

Development of an immunofluorescence assay module for determination of the mycotoxin zearalenone in water

Project Aquafluosense is designed to develop prototypes for a fluorescence-based instrumentation setup for in situ measurements of several characteristic parameters of water quality. In the scope of the project an enzyme-linked fluorescent immunoassay (ELFIA) method has been developed for the detection of several environmental xenobiotics, including mycotoxin zearalenone (ZON). ZON, produced by several plant pathogenic Fusarium species, has recently been identified as an emerging pollutant in surface water, presenting a hazard to aquatic ecosystems. Due to its physico-chemical properties, detection of ZON at low concentrations in surface water is a challenging task. The 96-well microplate-based fluorescence instrument is capable of detecting ZON in the concentration range of 0.09–400 ng/mL. The sensitivity and accuracy of the analytical method has been demonstrated by a comparative assessment with detection by high-performance liquid chromatography and by total internal reflection ellipsometry. The limit of detection of the method, 0.09 ng/mL, falls in the low range compared to the other reported immunoassays, but the main advantage of this ELFIA method is its efficacy in combined in situ applications for determination of various important water quality parameters detectable by induced fluorimerty—e.g., total organic carbon content, algal density or the level of other organic micropollutants detectable by immunofluorimetry. In addition, the immunofluorescence module can readily be expanded to other target analytes if proper antibodies are available for detection

A cytotoxic survey on 2-amino-1H-imidazol based synthetic marine sponge alkaloid analogues

Here, we describe the synthesis and biologic activity evaluation of 20 novel synthetic marine sponge alkaloid analogues with 2-amino-1H-imidazol (2-AI) core. Cytotoxicity was tested on murine 4T1 breast cancer, A549 human lung cancer, and HL-60 human myeloid leukemia cells by the resazurin assay. A total of 18 of 20 compounds showed cytotoxic effect on the cancer cell lines with different potential. Viability of healthy human fibroblasts and peripheral blood mononuclear cells upon treatment was less hampered compared to cancer cell lines supporting tumor cell specific cytotoxicity of our compounds. The most cytotoxic compounds resulted the following IC50 values 28: 2.91 µM on HL-60 cells, and 29: 3.1 µM on 4T1 cells. The A549 cells were less sensitive to the treatments with IC50 15 µM for both 28 and 29. Flow cytometry demonstrated the apoptotic effect of the most active seven compounds inducing phosphatidylserine exposure and sub-G1 fragmentation of nuclear DNA. Cell cycle arrest was also observed. Four compounds caused depolarization of the mitochondrial membrane potential as an early event of apoptosis. Two lead compounds inhibited tumor growth in vivo in the 4T1 triple negative breast cancer and A549 human lung adenocarcinoma xenograft models. Novel marine sponge alkaloid analogues are demonstrated as potential anticancer agents for further development