Le sostanze chimiche che alterano il sistema endocrino (EDCs) sono composti esogeni in grado di interferire con i meccanismi fisiologici di sintesi e segnalazione ormonale; i pesticidi ne sono inclusi, rappresentando un potenziale problema di salute pubblica. Lo scopo di questo progetto è quello di valutare se otto pesticidi, rilevati nelle acque superficiali e sotterrane piemontesi (2,6-diclorobenzamide, AMPA, azoxystrobin, imazamox, metolachlor, nicosulfuron, quinclorac, terbutilazin-desethyl), interferiscano con la sintesi e la segnalazione degli estrogeni: sono stati scelti come possibili target l'aromatasi e i recettori degli estrogeni (ERs) (in seguito a un MELN gene reporter assay, quinclorac e terbutilazina-desetil hanno mostrato attività estrogenica). L'aromatasi è un enzima chiave nella sintesi degli estrogeni, a partire dagli androgeni, attraverso l'aromatizzazione dell'anello A, costituendo così un bersaglio per gli EDCs. È stato eseguito un test di legame in spettroscopia per valutare il possibile legame dei pesticidi con l'aromatasi, i cui risultati hanno mostrato un legame di tipo I con l'AMPA e di tipo II con l'imazamox; 2,6-diclorobenzamide e nicosulfuron non instaurano interazione, mentre gli spettri dei restanti pesticidi sono stati influenzati dall'interferenza del solvente. Per indagare la possibile interferenza dei pesticidi con la conversione dell'androstenedione in estrone, catalizzata dall'aromatasi, è stato eseguito un test di attività in HPLC sia in assenza sia in presenza di pesticidi 10 μM: non è stato osservato alcun cambiamento significativo nell'attività dell'aromatasi. Il protocollo di preparazione della reazione è stato quindi ottimizzato e la concentrazione finale dei pesticidi aumentata a 50 μM: è stata rilevata un'inibizione dell'attività enzimatica da parte di quinclorac (46,2%), azossistrobina (13,9%), metolaclor (35,8%), imazamox (7%) e nicosulfuron (11,0%). Tuttavia, l'analisi dovrebbe essere approfondita ulteriormente per comprendere il loro meccanismo d'azione e con quale fase della reazione interferiscano. La linea cellulare di carcinoma colorettale HT29 è stata scelta per valutare gli effetti di quattro pesticidi: glifosato (inibitore dell'attività dell'aromatasi e probabile cancerogeno) e AMPA, quinclorac e terbutilazin-desethyl (con attività estrogenica dimostrata). Il saggio di vitalità con cristal violetto ha mostrato che AMPA e glifosato inducono un aumento significativo della vitalità cellulare dopo 48 e 72 ore di esposizione (5-100 μM); i risultati sono stati confermati dal saggio di proliferazione WST-1 dopo 72 ore di incubazione per concentrazioni di 5 e 50 μM. Il test è stato ripetuto in presenza di exemestano (inibitore dell'aromatasi) o tamoxifene (modulatore degli ERs) per indagare se l'effetto potesse essere correlato all'aromatasi e/o agli ERs. In presenza sia di pesticidi sia di exemestano, l'effetto è stato invertito (diminuzione della proliferazione), suggerendo un coinvolgimento significativo dell'aromatasi. Test preliminari sono stati eseguiti allo stesso modo con quinclorac e terbutilazin-desethyl, con conseguente diminuzione della vitalità cellulare in presenza dei pesticidi. Sono stati quindi identificati nuovi interferenti endocrini tra pesticidi diffusi nelle acque piemontesi che possono costituire un pericolo per la salute umana e ambientale: è necessario approfondire lo studio delle loro proprietà con l'obiettivo di promuoverne un uso più consapevole e rigoroso.
Endocrine disrupting chemicals (EDCs) are exogenous compounds able to interfere with physiological mechanisms of hormone synthesis and signalling; pollutants as pesticides are included, thus representing a potential public health concern. The aim of this project was to evaluate if eight selected pesticides detected in Piedmont surface and ground waters (2,6-dichlorobenzamide, AMPA, azoxystrobin, imazamox, metolachlor, nicosulfuron, quinclorac, terbuthylazin-desethyl) can interfere with estrogens synthesis and signalling; for this purpose, aromatase and estrogen receptors (ERs) were chosen as possible targets (ERs agonism and antagonism were previously assessed by MELN gene reporter assay, identifying ERs-mediated transcriptional activation by quinclorac and terbuthylazine-desethyl). Human aromatase (CYP19A1) is a key enzyme in estrogens synthesis, by catalysing conversion of androgens in estrogens through aromatisation of the A ring, therefore constituting a potential EDCs target. Firstly, a spectroscopic binding assay was performed to assess pesticides possible binding to aromatase, whose results showed type I binding by AMPA and type II by imazamox; 2,6-dichlorobenzamide and nicosulfuron can’t engage any type of interaction, while the obtained spectra of the remaining compounds of interest were affected by solvent interference. In order to further investigate a possible pesticides interference with conversion of androstenedione into estrone catalysed by aromatase, an activity assay was performed at HPLC both in absence and in presence of 10 μM pesticides: no significant change in aromatase activity was observed. Afterwards, reaction preparation protocol was optimised, and pesticides final concentration was increased to 50 μM: this set up allowed to detect an inhibition of aromatase activity by quinclorac (46.2%), azoxystrobin (13.9%), metolachlor (35.8%), imazamox (7%) and nicosulfuron (11.0%). However, analysis should be furthered to better inspect pesticides mechanism of action and with which reaction phase they can interfere. HT29 colorectal adenocarcinoma cell line was chosen to assess effects at cellular level of four selected pesticides: glyphosate (known inhibitor of aromatase activity and probable human carcinogen) and AMPA, quinclorac and terbuthylazin-desethyl (proven to exert estrogenic activity). Crystal violet viability assay showed that AMPA and glyphosate induced a significant increase in cell viability after 48 and 72 hours of exposure (in a concentration between 5 and 100 μM); these results were confirmed by WST-1 proliferation assays after 72-hours incubation for 5 and 50 μM final concentrations. The assay was repeated in presence of exemestane (aromatase inhibitor) or tamoxifen (ERs modulator) in order to investigate if the effect could be related to aromatase and/or ERs. In presence of both pesticide and exemestane, the effect was reverted (decrease in proliferation), thus suggesting a significant involvement of aromatase. Preliminary tests were performed in the same way with quinclorac and terbuthylazin-desethyl, resulting into a decrease in cell viability upon presence of the pesticide. Through this study, new endocrine disruptors have been identified among widespread pesticides in Piedmont waters, underlying the possible hazard they can constitute for human and wildlife health; therefore, research should go deeper into the investigation of their properties with the aim of promoting a more aware and strict use.
Identificazione dei pesticidi come nuovi interferenti endocrini: la loro interferenza con l'attività dell'aromatasi e con la vitalità delle cellule tumorali di colon HT29
ARMELLINO, FRANCESCA
2022/2023
Abstract
Endocrine disrupting chemicals (EDCs) are exogenous compounds able to interfere with physiological mechanisms of hormone synthesis and signalling; pollutants as pesticides are included, thus representing a potential public health concern. The aim of this project was to evaluate if eight selected pesticides detected in Piedmont surface and ground waters (2,6-dichlorobenzamide, AMPA, azoxystrobin, imazamox, metolachlor, nicosulfuron, quinclorac, terbuthylazin-desethyl) can interfere with estrogens synthesis and signalling; for this purpose, aromatase and estrogen receptors (ERs) were chosen as possible targets (ERs agonism and antagonism were previously assessed by MELN gene reporter assay, identifying ERs-mediated transcriptional activation by quinclorac and terbuthylazine-desethyl). Human aromatase (CYP19A1) is a key enzyme in estrogens synthesis, by catalysing conversion of androgens in estrogens through aromatisation of the A ring, therefore constituting a potential EDCs target. Firstly, a spectroscopic binding assay was performed to assess pesticides possible binding to aromatase, whose results showed type I binding by AMPA and type II by imazamox; 2,6-dichlorobenzamide and nicosulfuron can’t engage any type of interaction, while the obtained spectra of the remaining compounds of interest were affected by solvent interference. In order to further investigate a possible pesticides interference with conversion of androstenedione into estrone catalysed by aromatase, an activity assay was performed at HPLC both in absence and in presence of 10 μM pesticides: no significant change in aromatase activity was observed. Afterwards, reaction preparation protocol was optimised, and pesticides final concentration was increased to 50 μM: this set up allowed to detect an inhibition of aromatase activity by quinclorac (46.2%), azoxystrobin (13.9%), metolachlor (35.8%), imazamox (7%) and nicosulfuron (11.0%). However, analysis should be furthered to better inspect pesticides mechanism of action and with which reaction phase they can interfere. HT29 colorectal adenocarcinoma cell line was chosen to assess effects at cellular level of four selected pesticides: glyphosate (known inhibitor of aromatase activity and probable human carcinogen) and AMPA, quinclorac and terbuthylazin-desethyl (proven to exert estrogenic activity). Crystal violet viability assay showed that AMPA and glyphosate induced a significant increase in cell viability after 48 and 72 hours of exposure (in a concentration between 5 and 100 μM); these results were confirmed by WST-1 proliferation assays after 72-hours incubation for 5 and 50 μM final concentrations. The assay was repeated in presence of exemestane (aromatase inhibitor) or tamoxifen (ERs modulator) in order to investigate if the effect could be related to aromatase and/or ERs. In presence of both pesticide and exemestane, the effect was reverted (decrease in proliferation), thus suggesting a significant involvement of aromatase. Preliminary tests were performed in the same way with quinclorac and terbuthylazin-desethyl, resulting into a decrease in cell viability upon presence of the pesticide. Through this study, new endocrine disruptors have been identified among widespread pesticides in Piedmont waters, underlying the possible hazard they can constitute for human and wildlife health; therefore, research should go deeper into the investigation of their properties with the aim of promoting a more aware and strict use.| File | Dimensione | Formato | |
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https://hdl.handle.net/20.500.14240/147416