Endocrine disruptors are a compound category in which many pesticides have found place. They are highly hazardous for the organism, impacting the delicate balance of hormones involved in the regulation of a myriad of processes. Years of indiscriminate pesticides usage have left residues of these compounds in the soil, water and air we breathe, placing ourselves and the environment in danger. Amidst the wide variety of products and compounds, Glyphosate is one of the most distributed, utilised, and influential herbicides currently been used. Glyphosate and the formulations that utilise it as their active principle have an inhibitory effect on the shikimate pathway, leading to a slow death of the plant. Given its short half-life and the fact that other organisms lack its target enzyme (5-enolpyruvylshikimate-3-phosphate synthase), Glyphosate has been considered to be a beneficial molecule for many decades. Nevertheless, besides Glyphosate’s dozens of applications and field of usage, the toxic effects of the compound have long been the subject of debate. Studies have reconducted exposure to sub-toxic Glyphosate concentrations to a plethora of conditions. Epidemiological studies have found many associations between the compound and cancer insurgency. Moreover, disruption of the endocrine system is one of the most cited effects caused by Glyphosate. Various health organisations have all taken different positions on Glyphosate safety and its cancerogenic profile, turning it into a very controversial topic. In this thesis work, I investigated whether Glyphosate could induce any detrimental effect on thyroid cells. This study in which I took part was among the first in vitro experiments to take place. Fisher-rat-thyroid-line-5; FRTL-5 cells were used and exposed to various non-cytotoxic Glyphosate concentrations for different time periods. These concentrations were chosen based on the existing literature. We evaluated the Glyphosate effect over adherent cellular colonies, and a reduction in both their viability and proliferation was noticed. The FRTL-5 cells were exposed to Glyphosate and utilised H2DCFDA combined with spectrophotometry techniques, observing an increase in ROS production occurring in cells exposed to at least 2 mM of the compound. We tested Glyphosate effect on the same cellular line seeded to produce 3D spheroid colonies. Spheres formation was impaired only at 10 mM Glyphosate concentration and a test confirmed the cause to be a reduction of cellular viability. Viability, proliferation and ROS production results obtained in 2D culture were confirmed by the 3D model. Lastly, we investigated the possible effect of Glyphosate on the expression of genes relevant for thyroid hormone synthesis including tshr, nis, tg, tpo, pax-8 and ttf1, for both the 2D and 3D cell model was investigated. RT-PCR allowed the quantification of transcripts of each gene, showing how the compound had influenced adherent and spheroid colonies in different ways. Glyphosate exposure generally increased the transcription for the majority of the genes. Considering all the observed results, this project shows the adverse cytotoxic effects of Glyphosate on thyroid cells.
Valutazione dell'esposizione al Glifosato in cellule tiroidee in vitro I distruttori endocrini sono composti altamente pericolosi per l'organismo, in quanto incidono sul delicato equilibrio ormonale, coinvolto nella regolazione di una miriade di processi. Molti pesticidi sono stati riconosciuti in questa categoria di composi. Anni di uso sconsiderato hanno lasciato e lasciano tuttora residui nel suolo, nell'acqua e nell'aria che respiriamo, esponendo noi e gli ecosistemi ad un silenzioso pericolo. Tra l'ampia varietà di prodotti e composti utilizzati, il Glifosato è uno degli erbicidi più diffusi e influenti. Il Glifosato e le formulazioni che lo includono come principio attivo hanno un effetto inibitorio nella via metabolica dello “shikimato” presente solo nelle piante, portando ad una lenta morte delle cosiddette piante infestanti. Data la breve emivita e la mancanza in organismi secondari dell’enzima bersaglio 3-fosfoshikimato 1-carbossiviniltransferasi (EPSPS) in altri, il Glifosato è stato considerato a lungo una molecola sicura. Tuttavia, i potenziali effetti tossici del composto sono stati a lungo oggetto di dibattito. Studi epidemiologici hanno trovato molte associazioni tra il composto e l'insorgenza di neoplasie. La sua azione di potenziale interferente endocrino è uno degli effetti più citati nell’attuale letteratura. Varie organizzazioni hanno assunto posizioni diverse in merito alla sicurezza del Glifosato e sul suo profilo cancerogeno, rendendo l’argomento della sua potenziale pericolosità molto controverso. In questo lavoro di tesi, ho valutato se il composto potesse indurre effetti dannosi sulle cellule della tiroide. Lo studio a cui ho partecipato è stato tra i primi eseguiti in vitro per quanto riguarda questo argomento. Le cellule che abbiamo utilizzato appartengono alla linea delle Fisher-rat-thyroid-line-5; FRTL-5. Queste sono state esposte per diversi periodi di tempo a varie concentrazioni di Glifosato, le quali sono state selezionate in base alla letteratura esistente. Abbiamo valutato l'effetto del Glifosato sulle cellule aderenti e abbiamo notato una riduzione della loro vitalità e proliferazione. In seguito, nelle cellule esposte, con l’aggiunta di H2DCFDA e in combinazione a tecniche di spettrofotometria, abbiano notato un aumento della produzione di ROS in presenza di almeno 2 mM del pesticida. Gli effetti del Glifosato sono stati testati parallelamente sulla stessa linea cellulare disposta in modello sferoide (3D). Questi sono stati esposti al pesticida durante i quattro giorni del processo di sferificazione. La formazione delle sfere è stata compromessa solo dalla concentrazione di Glifosato 10 mM. I risultati ottenuti sulle cellule aderenti per quanto riguarda viabilità e proliferazione e produzione di ROS sono stati confermati nel modello 3D. Infine su entrambi i modelli, abbiamo studiato il possibile effetto che il Glifosato potesse avere sull'espressione di geni coinvolti nella funzionalità tiroidea: TPO, TG, TSHR, PAX-8, TTF1 e NIS. La RT-PCR ci ha permesso di quantificare la trascrizione di ciascun gene, mostrando come il composto abbia avuto una diversa influenza in colonie aderenti e sferoidi, ma generalmente incrementando la trascrizione nella maggior parte di essi. Considerando tutti i risultati, questo progetto mostra effetti avversi del Glifosato sulle cellule tiroidee.
In vitro evaluation of exposure to Glyphosate in thyroid cells
MARCANTOGNINI, ANDREA
2021/2022
Abstract
Endocrine disruptors are a compound category in which many pesticides have found place. They are highly hazardous for the organism, impacting the delicate balance of hormones involved in the regulation of a myriad of processes. Years of indiscriminate pesticides usage have left residues of these compounds in the soil, water and air we breathe, placing ourselves and the environment in danger. Amidst the wide variety of products and compounds, Glyphosate is one of the most distributed, utilised, and influential herbicides currently been used. Glyphosate and the formulations that utilise it as their active principle have an inhibitory effect on the shikimate pathway, leading to a slow death of the plant. Given its short half-life and the fact that other organisms lack its target enzyme (5-enolpyruvylshikimate-3-phosphate synthase), Glyphosate has been considered to be a beneficial molecule for many decades. Nevertheless, besides Glyphosate’s dozens of applications and field of usage, the toxic effects of the compound have long been the subject of debate. Studies have reconducted exposure to sub-toxic Glyphosate concentrations to a plethora of conditions. Epidemiological studies have found many associations between the compound and cancer insurgency. Moreover, disruption of the endocrine system is one of the most cited effects caused by Glyphosate. Various health organisations have all taken different positions on Glyphosate safety and its cancerogenic profile, turning it into a very controversial topic. In this thesis work, I investigated whether Glyphosate could induce any detrimental effect on thyroid cells. This study in which I took part was among the first in vitro experiments to take place. Fisher-rat-thyroid-line-5; FRTL-5 cells were used and exposed to various non-cytotoxic Glyphosate concentrations for different time periods. These concentrations were chosen based on the existing literature. We evaluated the Glyphosate effect over adherent cellular colonies, and a reduction in both their viability and proliferation was noticed. The FRTL-5 cells were exposed to Glyphosate and utilised H2DCFDA combined with spectrophotometry techniques, observing an increase in ROS production occurring in cells exposed to at least 2 mM of the compound. We tested Glyphosate effect on the same cellular line seeded to produce 3D spheroid colonies. Spheres formation was impaired only at 10 mM Glyphosate concentration and a test confirmed the cause to be a reduction of cellular viability. Viability, proliferation and ROS production results obtained in 2D culture were confirmed by the 3D model. Lastly, we investigated the possible effect of Glyphosate on the expression of genes relevant for thyroid hormone synthesis including tshr, nis, tg, tpo, pax-8 and ttf1, for both the 2D and 3D cell model was investigated. RT-PCR allowed the quantification of transcripts of each gene, showing how the compound had influenced adherent and spheroid colonies in different ways. Glyphosate exposure generally increased the transcription for the majority of the genes. Considering all the observed results, this project shows the adverse cytotoxic effects of Glyphosate on thyroid cells.È consentito all'utente scaricare e condividere i documenti disponibili a testo pieno in UNITESI UNIPV nel rispetto della licenza Creative Commons del tipo CC BY NC ND.
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https://hdl.handle.net/20.500.14239/15461