Metabolomics approaches to understand cellular toxicity of glyphosate and a novel microchip-based detection method

Glyphosate, N-(phosphonomethyl)glycine, is the active compound in glyphosate based herbicides. It is currently the most intensely applied herbicide across the globe. Likewise, the potential harmfulness of glyphosate has been heatedly debated as some studies have linked glyphosate exposure to cancer development and endocrine disruption in humans and other life forms. This work first aimed to investigate the effects of glyphosate and a glyphosate-based herbicide (Roundup) on the metabolism of Arabidopsis thaliana plants by utilising an untargeted metabolomics approach. Secondly, pure glyphosate toxicity was inspected upon exposure of HEK 293 cells for up to 24 h. Lastly, due to the prevalence of glyphosate in non-targeted environments and its controversial toxicological status, a novel quantification method for glyphosate was developed. The untargeted metabolomics approach in Arabidopsis thaliana plants treated with both pure glyphosate and Roundup for up to 48 h confirmed the previously established mode of action of the herbicide. The shikimate pathway was targeted which led to an accumulation of metabolites upstream the 5- enolpyruvylshikimate-3-phosphate synthase inhibition point. Levels of aromatic amino acids, however, accumulated over time despite the shikimate pathway being disrupted. Metabolites involved in plant immunity were also observed to accumulate alongside levels of amino acids. The observed metabolic disruptions indicated that nutrient recycling and proteolysis were engaged possibly to ensure plant survival. Next, the untargeted metabolomics approach was also employed to study the effects of pure glyphosate on the metabolism of HEK 293 cells. The cells were exposed to an agriculturally relevant concentration of glyphosate (21 mM) for up to 24 h in serum-free medium. Interestingly, the analysis revealed that the purine and pyrimidin pathways were disrupted which are involved in DNA and RNA synthesis. Also, the metabolites resulting from glyphosate metabolism (AMPA and methylphosphonate) were detected. Lastly, a sensitive and fast optical-based detection method for underivatized glyphosate in water was devised. A portable, in-house produced glyphosate specific-enzyme was utilised which was paired with a complementary metal oxide semiconductor (CMOS) photodiode array sensor. The powerful 16 x 16 photodiode array chip enabled the optical real-time detection of glyphosate. The sensitivity for glyphosate quantification (LOD= 0.47 μM and LOQ= 1.55 μM) met the set standards by the WHO and the EPA in the U.S.. What is more, the enzymatic PD CMOS sensor was successful in quantifying glyphosate in spiked tap water samples and registered recoveries from 100-127 %