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Two commercially available ▫$TiO_2$▫ (hexagonal-like and spherical-like particles) were used to investigate the effect of ▫$g-C_3N_4$▫ “melting” on the photocatalytic properties of ▫$g-C_3N_4/TiO_2$▫ composites. Improvement in the contact between the components was observed when they were thermally treated at 350 °C for an extended period of time (between 2 and 72 h) due to the partial melting and phase fusion of ▫$g-C_3N_4$▫. Consequently, the enhanced contact between the phases allows easier injection of photogenerated electrons from the conduction band of ▫$g-C_3N_4$▫ into ▫$TiO_2$▫, improving charge carrier separation. The prepared composites were tested for bisphenol A degradation under visible-light illumination, which showed that the components that had been calcined for 24 h performed better due to the improved charge carrier separation. Superoxide anionic radicals and photogenerated holes were identified as active species in the photooxidation experiments conducted under visible-light illumination. Nasl. z nasl. zaslona. Soavtorji: Gregor Žerjav, Matjaž Finšgar, Janez Zavašnik, Albin Pintar. Opis vira z dne 13. 3. 2023. Bibliografija: str. 14-16. Abstract.

Dissertação de mestrado em Genética Molecular, Tetraselmis chuii is a species of microalgae with high nutritional value and its interest in nutraceutical applications is growing. In addition, is a rich source of a variety of bioactive compounds. Recognizing the biological potential of this species, the objective of this work was to test five different microalga biomasses produced by different extraction methods (with or without high pressure process HPP) and drying methods (drying by lyophilization and spray drying), regarding their antioxidant, antifungal and antigenotoxic activity. Antioxidant assays were carried out using different colorimetric biochemical tests (DPPH radical scavenging activity, metal ion chelating assay, ferric ion reducing antioxidant power and superoxide and nitric oxide scavenging activity). Antifungal activity was tested by the agar diffusion method against the phytopathogenic fungal species Colletotrichum nymphaea, Phytophthora cinnamomi, Botrytis cinerea and Diplodia corticola. The antigenotoxic activity was evaluated using the genotoxic agents camptothecin, hydrogen peroxide and bisphenol A and the yeast Schizosaccharomyces pombe as a model organism. T. chuii biomasses showed antioxidant activity with higher values for iron chelation capacity (47%- 94% of activity) and superoxide radical scavenging (62%-75% of activity). Regarding the antifungal capacity, only two of the tested biomasses (deconstructed biomass HPP and integral biomass) showed the capacity to inhibit the growth of the fungal species C. nymphaea, P. cinnamomi and B. cinerea. Finally, all T. chuii biomasses were able to mitigate the genotoxic effects (morphological changes and cell cycle delay) of bisphenol A in yeast cells. Comparing the bioactivity of different biomasses, the drying process seems to negatively affect the antioxidant and antifungal activity, on the other hand, the application of the high-pressure method varies according to the tests. These differences do not occur in antigenotoxic activity, all biomasses oppose the effect of bisphenol A in the same way. T. chuii proves to be a very promising microalga for future uses in biotechnology., Tetraselmis chuii é uma espécie de microalga com alto valor nutricional e o interesse desta para aplicações nutracêuticas é crescente. Além disso, é uma fonte rica de uma variedade de compostos bioativos. Reconhecendo o potencial biológico desta espécie, o objetivo deste trabalho foi testar cinco biomassas de microalga produzidas através de diferentes métodos de extração (com ou sem processo de alta pressão HPP) e métodos de secagem (secagem por liofilização e “spray-drying”) quanto à atividade antioxidante, antifúngica e antigenotóxica. Os ensaios antioxidantes foram realizados através de testes bioquímicos colorimétricos (atividade de eliminação de radicais DPPH, ensaio de quelação de iões férricos, capacidade redutora de iões férricos e atividade sequestradora de superóxido e óxido nítrico). A atividade antifúngica foi testada usando método de difusão em agar contra as espécies de fungos fitopatogénicos Colletotrichum nymphaea, Phytophthora cinnamomi, Botrytis cinerea e Diplodia corticola. A atividade antigenotóxica foi avaliada utilizando os agentes genotóxicos camptotecina, peróxido de hidrogénio e bisfenol A e a levedura Schizosaccharomyces pombe como organismo modelo. T. chuii apresentou valores mais elevados de atividade antioxidante para a capacidade de quelação de ferro (47%-94% de atividade) e de interceção do radical superóxido (62%-75% de atividade). Relativamente à capacidade antifúngica, apenas duas das biomassas testadas (biomassa desconstruída HPP e biomassa integral) apresentaram capacidade de inibir o crescimento das espécies de fungos C. nymphaea, P. cinnamomi e B. cinerea. Por último, todas as biomassas de T. chuii apresentaram capacidade de mitigar os efeitos genotóxicos (alterações morfológicas e atraso no ciclo celular) do bisfenol A em células de levedura. Comparando a bioatividade das diferentes biomassas, o processo de secagem parece afetar negativamente a atividade antioxidante e antifúngica, por outro lado, a aplicação do método de alta pressão varia de acordo com os ensaios. Estas diferenças não são obsevadas na atividade antigenotóxica, todas as biomassas contrariam o efeito do bisfenol A de igual forma. T. chuii revela ser uma microalga bastante promissora para futuras utilizações na biotecnologia.

Los contaminantes emergentes (CE) representan un riesgo significativo para la salud humana, ecológica y ambiental. En los últimos años, la ciencia ha avanzado en el estudio de los efectos sobre la fauna acuática. Sin embargo, los efectos de estos sobre la salud humana aún no son reconocidos. Por lo cual legislación y el control de los CE aun es materia de discusión. No obstante, se detectan de manera ubicua en la hidrosfera, el suelo y la biota, donde las principales vías de entrada al ambiente se han relacionado principalmente con la excreción y eliminación de productos farmacéuticos y de cuidado personal (PPCP), la inadecuada disposición de residuos veterinarios, los lixiviados de rellenos sanitarios y la poca remoción de estos en las plantas de tratamiento de aguas residuales. Los CE se encuentran tan ampliamente distribuidos que se pueden encontrar en las aguas subterráneas, comprometiendo su calidad e inocuidad. En la región de La Mojana, ubicada en el norte de Colombia, habitan aproximadamente 450.461 personas, que en su mayoría dependen del agua subterránea para satisfacer sus necesidades básicas y para la explotación agrícola (Conpes 4084., 2022). Aunque en esta región se han realizado estudios sobre CE en aguas superficiales, con respecto a las subterráneas no se tienen registros, por lo que en este estudio se planteó como objetivo identificar la presencia de los CE cafeína (CAF), Gemfibrozilo (GEM), 17- Alfa-etinilestradiol (EE2) y bisfenol A (BPA), en el agua subterránea de La Mojana, mediante cromatografía líquida de alta eficiencia con detector de arreglo de diodos y fluorescencia molecular (HPLC-DAD-FLD) y confirmación por cromatografía de gases con espectrómetro de masas (GC/MS). Para este estudio se seleccionaron 20 sitios de muestreo distribuidos en los municipios de San Marcos, Majagual, La Unión, San Benito de Abad, Guaranda, Caimito y Sucre. Donde no se encontró presencia de GEM, EE2 y CAF en ninguno de los sitios evaluados. No obstante, el BPA se encontró en un 70% de las muestras analizadas, en el intervalo de concentraciones de 0,07 – 0,410 μg/L. Evidenciando la contaminación de las aguas subterráneas de la región de La Mojana con CE. 1 INTRODUCCIÓN ..................................................................................................................2 2 MARCO TEÓRICO.................................................................................................................4 2.1 ANTECEDENTES ............................................................................................................4 2.2 CONTAMINANTES EMERGENTES...............................................................................9 2.3 PRODUCTOS FARMACEUTICOS Y DE CUIDADO PERSONAL (PPCP)....................9 2.3.1 DISRUPTORES ENDOCRINOS (EDC)...................................................................10 2.3.1 DISRUPTORES ENDOCRINOS (EDC)...................................................................10 2.3.2 VIAS DE ENTRADA DE LOS CEs EN EL AMBIENTE.........................................12 2.3.3 BISFENOL A (BPA).................................................................................................13 2.3.4 17-ALFA-ETINILESTRADIOL (EE2) ....................................................................15 2.3.5 CAFEÍNA (CAF)......................................................................................................17 2.3.6 GEMFIBROZILO (GEM).........................................................................................19 2.3.7 TÉCNICA ANALÍTICA ...........................................................................................20 2.3.8 CROMATOGRAFÍA DE LÍQUIDOS EN FASE REVERSA (RP-HPLC).................21 2.3.9 DETECTOR DE AREGLO DE DIODOS DAD ........................................................21 2.3.10 DETECTOR DE FLUORESCENCIA (FLD) ..........................................................22 2.3.11 EXTRACCIÓN EN FASE SÓLIDA (SPE) .............................................................23 3 OBJETIVOS..........................................................................................................................2 3.1 OBJETIVO GENERAL ...............................................................................................25 3.2 OBJETIVOS ESPECIFICOS .......................................................................................25 4 METODOLOGÍA ..................................................................................................................26 4.1 ÁREA DE ESTUDIO ......................................................................................................26 4.2 COLECTA DE MUESTRAS...........................................................................................26 4.3 ANÁLISIS DE PARÁMETROS FISICOQUÍMICOS......................................................28 4.4 EXTRACCIÓN Y LIMPIEZA.........................................................................................29 4.5 DETERMINACIÓN POR CROMATOGRÁFIA DE LÍQUIDOS....................................29 4.6 DETERMINACIÓN POR CROMATOGRÁFIA DE GASES..........................................30 4.7 CONTROL DE CALIDAD ANALÍTICO........................................................................30 4.8 ANÁLISIS ESTADÍSTICO.............................................................................................31 5 REACTIVOS, EQUIPOS Y MATERIALES..........................................................................31 6 RESULTADOS Y DISCUSIÓN ............................................................................................32 6.1 VARIABLES FISICOQUÍMICAS ..................................................................................33 6.2 CONCENTRACIÓN DE CEs..........................................................................................37 6.3 CORRELACIÓN DE LOS PARÁMETROS FISICOQUÍMICOS Y LAS CONCENTRACIONES DE CEs...........................................................................................42 7 CONCLUSIONES .................................................................................................................46 8 RECOMENDACIONES ........................................................................................................47 9 BIBLIOGRAFIA ..................................................................................................................48 10 ANEXOS .............................................................................................................................58 10.1 Anexo 1: TOMA DE MUESTRAS ................................................................................58 10.2 Anexo 2: MEDICIÓN DE PARAMETROS FISICOQUIMICOS...................................60 10.3 Anexo 3: EXTRACCIÓN Y LIMPIEZA........................................................................61 10.4 Anexo 4: CONTROL DE CALIDAD.............................................................................61 10.4.1 Rectas de calibrado..................................................................................................61 10.4.2 Porcentajes de recuperación en muestras fortificadas...............................................62 10.5 Anexo 5: CROMATOGRAMAS ...................................................................................63 10.6 Anexo 7: POZOS ..........................................................................................................69 Pregrado Químico(a) Trabajos de Investigación y/o Extensión

Bisfenol A (BPA) je že vrsto let znan motilec endokrinega sistema. V zadnjih letih narašča ozaveščenost o njegovih negativnih vplivih na zdravje, kar je botrovalo zaostritvi zakonodaje glede njegove uporabe. To je vzrok za razvoj in uporabo analogov BPA, ki so novejše spojine s podobno kemijsko strukturo BPA. Novejše študije BPA pripisujejo tudi imunomodulatorne lastnosti, prav tako pa njegovim analogom, kar smo preverili v našem magistrskem delu. Preučevali smo vpliv devetih analogov BPA na modulacijo sedmih citokinov v treh limfoblastoidnih celičnih linijah (LCL). Najprej smo s testom metabolne aktivnosti MTS določili, kako izbrani bisfenoli vplivajo na živost celic. V ta namen smo določili srednjo inhibitorno koncentracijo (IC50), pri kateri je bilo presnovno aktivnih še 50 % celic. Določene vrednosti IC50 so se med bisfenoli precej razlikovale. Tako smo najnižjo povprečno vrednost IC50 in s tem največjo citotoksičnost za to vrsto celic določili naslednjim analogom: BPP (IC50 =27,9 μM) , BPAF (IC50 = 52,6 μM), BPAP (IC50 =57,7 μM) in BPB (IC50 =97,7 μM). Nekoliko manjšo citotoksičnost smo določili BPA (IC50 =175,5 μM), ostali preučevani analogi (BPE, 2,4-BPS, BPF in TMBPF) pa so imeli višje vrednosti IC50, ki so presegle najvišjo testirano, 200 μM koncentracijo. Ugotovili smo, da je vrednost IC50 obratno sorazmerna z vrednostjo logP, ki pa je direktno povezan z lipofilnostjo. Korelacija je bila visoka, torej gre za močno povezanost med lipofilnostjo in citotoksičnostjo. Nato smo določili vpliv izbranih analogov BPA na modulacijo sedmih citokinov. Celice LCL smo izpostavili trem različnim koncentracijam izbranih bisfenolov, 10 μM, 100 nM in 10 nM, jih aktivirali s kombinacijo ionomicina in PMA ter na pretočnem citometru ovrednotili vpliv na sproščanje citokinov. Pri 10 μM so bisfenoli povzročili znižanje večine merjenih citokinov, in sicer tako vnetnih citokinov IL-2, IL-6 in TNF-α kot tudi imuno-regulatornega citokina IL-10, iz česar lahko sklepamo na njihovo imunosupresivno delovanje pri visokih koncentracijah. Pri fiziološko možno prisotnih 100 nM in 10 nM koncentracijah pa so bisfenoli delovali pro-vnetno, saj so povečali koncentracijo vnetnega citokina IL-2 in pleiotropnega IFN-γ ter zmanjšali koncentracijo imuno-regulatornega citokina IL-10. Iz tega lahko sklepamo, da učinek BPA in njegovih analogov na izločanje citokinov ni linearno odvisen od njihove koncentracije, kar nakazujejo tudi predhodne študije. Iz rezultatov magistrskega dela lahko zaključimo, da imajo analogi BPA imunomodulatorno delovanje, saj vplivajo na izločanje citokinov v celicah LCL. Smiselne bi bile dodatne študije, ki bi razjasnile vpliv analogov BPA na modulacijo imunskega sistema. Bisphenol A (BPA) has been known for many years as endocrine disrupting chemical. In recent years, awareness of its negative effects on health has increased, which has encouraged the tightening of regulations regarding its use. This is the reason for the development and use of BPA analogues, which are newer compounds with a similar chemical structure to BPA. Recent studies also attribute immunomodulatory properties to BPA, as well as to its analogue, which we verified in our master's thesis. We studied the effect of nine BPA analogs on modulation of seven cytokines in three lymphoblastoid cell lines (LCL). First, we used the MTS cell viability assay to determine the effect of selected bisphenols on cell viability. For this purpose, we determined the half inhibitory concentration (IC50), at which 50 % of the cells were still metabolically active. The determined IC50 values differed significantly among the bisphenols. The lowest average IC50 value and thus the highest cytotoxicity for this type of cells was determined by the following analogues: BPP (IC50 = 27,9 μM), BPAF (IC50 = 52,6 μM), BPAP (IC50 =57,7 μM) and BPB (IC50 =97,7 μM). Lower cytotoxicity was determined for BPA (IC50 = 175,5 μM), while the other studied analogues (BPE, 2,4-BPS, BPF and TMBPF) had higher IC50 values that exceeded the highest tested concentration of 200 μM. We found that the IC50 value is inversely proportional to the logP value, which is directly related to lipophilicity. The correlation was high, so there is a strong correlation between lipophilicity and cytotoxicity. Next, we determined the influence of selected BPA analogues on the modulation of seven cytokines. LCL cells were exposed to three different concentrations of selected bisphenols, 10 μM, 100 nM and 10 nM, activated with a combination of ionomycin and PMA, and then we evaluated the effect on release of cytokines by a flow cytometry. At 10 μM bisphenols caused a decrease in most of the measured cytokines, specifically the pro-inflammatory cytokines IL-2, IL-6 and TNF-α as well as the immuno-regulatory cytokine IL-10, which indicate their immunosuppressive effects at high concentrations. At the physiologically possible concentrations of 100 nM and 10 nM, bisphenols had a pro-inflammatory effect, as they increased the concentration of the pro-inflammatory cytokine IL-2 and the pleiotropic IFN-γ and decreased the concentration of the immuno-regulatory cytokine IL-10. Results indicate that the immunomodulatory effects of BPA and its analogues on cytokine production are not concentration-dependent, which is also in line with previous studies. From the results of this Master's Thesis, we can conclude that BPA analogues have an immunomodulatory effect and otherwise affect the secretion of cytokines in LCL cells. Additional studies examining the impact of BPA analogs on immune system modulation would be worthwhile.

Dissertação para obtenção do grau de Mestre no Instituto Universitário Egas Moniz O bisfenol A (BPA) é um dos produtos químicos de maior volume produzidos em todo o mundo. Ele é utilizado, por exemplo, na produção de resinas epóxi e de plásticos de policarbonato os quais que se encontram em muitos produtos de consumo. Vários estudos, realizados em humanos, mostraram níveis mensuráveis de BPA nos fluídos corporais, sendo encontrado em adultos, crianças e também grávidas. Com uma estrutura muito semelhante à dos estrogénios, ele foi classificado como um desregulador endócrino. Por causa da sua presença, vários efeitos foram observados, desde alterações comportamentais, cardiovasculares, no aparelho reprodutor, e até na cavidade oral. Na área da medicina dentária, os monómeros mais encontrados são a família dos di-metacrilatos da qual o bis-GMA e seus derivados são mais usados. Presentes maioritariamente na parte orgânica das resinas compostas também se pode encontrar em quantidade inferior na parte resinosa dos adesivos. Precauções, por parte do médico dentista, podem ser tomadas para reduzir grande parte da libertação do BPA após colocação do material restaurador no paciente. No entanto, novas alternativas estão presentes no mercado, que resolvem à partida a questão dos desreguladores endócrinos por não conter derivados de BPA nas suas composições. Essas alternativas, revelam propriedades promissoras. Bisphenol A (BPA) is one of the highest volume chemicals produced worldwide. It is used, for example, in the production of epoxy resins and polycarbonate plastics which are found in many consumer products. Several human studies have shown measurable levels of BPA in body fluids. Found in adults, children, and also pregnant women. With a structure very similar to the estrogens, it has been classified as an endocrine disruptor. Because of its presence, several effects were observed, from behavioral changes, cardiovascular, reproductive system, even in the oral cavity. In the area of dentistry, the most common monomers are the family of dimethacrylates from which bis-GMA and its derivatives are most often used. Present mainly in the organic part of composite resins, they can also be found in smaller quantities in the resinous part of adhesives. The dentist can take some precautions to reduce much of the BPA release after placement of the restorative material in the patient. However, new alternatives are on the market, which resolve the issue of endocrine disruptors in the first place, as they do not contain BPA derivatives in their compositions. These alternatives reveal promising properties.

Bisfenol A (BPA) günlük hayatta sıklıkla maruz kalınan, çevresel olarak çok yaygın bulunduğu tespit edilen bir bileşiktir. BPA insan maruziyeti çok sık görülen endokrin bozucu bir kimyasaldır. Bu maddenin bilinçsizce kullanımının ekosistem ve halk sağlığı üzerinde olumsuz etkileri çoğu araştırmaya konu olmuştur. Bu çalışma BPA'nın neden olduğu oksidatif strese maruz kalan ratlarda karvakrolün olası koruyucu etkisini araştırmayı amaçladı. Çalışmada toplam 42 adet Wistar Albino rat kullanıldı. Çalışma süresi 30 gün sürdü. Deney hayvanlarına BPA ve karvakrol gastrik gavaj yoluyla verildi. 1. Grup negatif kontrol grubu olarak belirlendi. 2. Grup yağ grubu olarak belirlendi. 3. Grup pozitif kontrol grubu olarak belirlendi. 3., 4., 5., 6. Gruplara günde bir defa 25 mg/kg BPA verildi. Ayrıca, 4.5.ve 6. gruplara sırasıyla 12,5, 25 ve 50 mg/kg karvakrol günde bir defa verildi. Alınan dokularda oksidatif stres parametreleri (SOD ve CAT aktivite düzeyleri, MDA ve GSH seviyeleri) patolojik analizleri ve moleküler analizleri (IFN-γ, NFκB ve TNF-α proinflamatuvar genleri) yapıldı. BPA uygulamasıyla antioksidan enzimlerin aktivite düzeyleri ve glutatyon düzeylerinde azalma görülürken lipid peroksidasyon seviyelerinde artış görüldü. Dokularda histopatolojik değişiklikler ve proinflamatuvar genlerin ekspreyon seviyelerinde yükselme ve gözlemlendi. Karvakrol uygulamasının bu olumsuz etkileri tersine çevirdiği görüldü. Çalışmanın sonucunda BPA ile meydana getirilen oksidatif strese karşı karvakrolün doğal antioksidan olarak koruyucu etkisinin olduğu belirlendi. Bisphenol A (BPA) is a compound that is frequently exposed in daily life and found to be very common in the environment. BPA is an endocrine-disrupting chemical that is very common in human exposure. The negative effects of the inappropriate use of this substance on ecosystem and public health were issued in many studies. This study aimed to investigate the possible protective effect of carvacrol in rats exposed to oxidative stress caused by BPA. A total of 42 Wistar Albino rats were employed in the study. The working period lasted 30 days. BPA and carvacrol were given to the experimental animals by gastric gavage. Group 1 was served as the negative control group. The second group was served as the fat group. Group 3 was determined as the positive control group. Groups 3, 4, 5, and 6 were given 25 mg/kg BPA once a day. In addition, 12.5 , 25 and 50 mg/kg carvacrol were given once a day to groups 4.5 and 6, respectively. Oxidative stress parameters (SOD and CAT activity levels, MDA and GSH levels) pathological analyzes and molecular analyzes (IFN-γ, NFκB and TNF-α proinflammatory genes) were performed in the obtained tissues. With BPA application, the activity levels of antioxidant enzymes and glutathione levels decreased, while lipid peroxidation levels increased. Histopathological alterations in tissues and increased expression levels of proinflammatory genes were observed. It was shown that the administration of carvacrol reversed these negative effects. Consequently, it was determined that carvacrol has a protective effect as a natural antioxidant against oxidative stress caused by BPA. Bu tez çalışması; Afyon Kocatepe Üniversitesi Bilimsel Araştırma Projeleri Koordinasyon Birimi (BAPK) Tarafından Desteklenmiştir. Proje No:“21.SAĞ.BİL.32”

V doktorski disertaciji smo se osredotočili na identifikacijo potencialnih hormonskih motilcev fenolnih analogov bisfenola A (BPA) iz skupin zdravil, onesnažil in kozmetičnih sestavin, na njihovo pleiotropno delovanje ter preučili hormonske učinke njihovih mešanic. Najprej smo preverili zastavljeno hipotezo doktorske disertacije ali je modulacija hormonskega sistema odvisna od kemijske strukture. Med fenolnimi analogi BPA smo identificirali prepoznavne strukturne elemente odgovorne za interakcijo z jedrnimi receptorji.Z metodo in silico smo s pomočjo prosto dostopnega programskega paketa Endocrine Disruptome smo napovedali afiniteto vezave z 12 različnimi jedrnimi receptorji za izbran niz spojin. Predvsem smo se osredotočili na bromirana zaviralca gorenja TBB in TBPH, njunametabolita (TBBA in TBMEPH) ter aminofenolne analoge BPA (diklofenak (DIC), 4- hidroksidiklofenak (4-HD), paracetamol (PAR) in piceatanol (PIC)). Testirali smo jih in vitro na celičnih linijah s poročevalskim genom za luciferazo: MDA-kb2 celični liniji, ki ima izražen glukokortikoidni (GR) kot tudi androgeni receptor (AR), in GH3.TRE-Luc celični liniji z izraženima tiroidnima receptorjema α in β (ΤRα in TRβ). Za spojine, ki so izkazovale glukokortikoidno in androgeno moduliranje, smo izvedli še potrditveni test vezave in vitro. Med prvimi smo za prej omenjene spojine dokazali njihove različne hormonske učinke na estrogenskem (ER), androgenem, glukokortikoidnem in tiroidnem receptorju pri koncentracijah, katerim smo ljudje realno lahko izpostavljeni. Ugotovili smo, da metabolizem neposredno vpliva na modulacijo androgenega, glukokortikoidnega in tiroidnega receptorja. Za modulacijo glukokortikoidnega receptorja so pomembni: prosta karboksilna skupina in 2-(etilheksil)-oksikarbonilna skupina vezani na aromatski obroč ter prisotnost treh halogenih atomov v orto položaju. Rezultati so pokazali, da je antiandrogena aktivnost odvisna od števila karbonilnih skupin v molekuli in stilbenske strukture brez dodatnih stranskih verig med benzenskima skupinama. Ugotovili smo, da afiniteto vezave na androgeni receptor izkazujejo spojine, ki imajo v svoji strukturi Ph-N-C=O fragment, OH in NH-C=O skupini ter aromatski obroč. Na splošno, spojine, ki so bromirane na položaju 3 in 5 na fenilnem obroču glede na osnovni skelet, imajo antitiroidni učinek. Opazili smo, da je Ph-N-Ph fragment, ki vsebuje še atom Cl na enem od fenilnih obročev, prisoten pri spojinah z mešanim agonističnim/antagonističnim delovanjem na androgeni receptor. Ugotovili smo tudi, da hormonski motilci androgenega, glukokortikoidnega, tiroidnega in estrogenega receptorja v svoji strukturi vsebujejo fenolne skupine kot tudi OH skupino v para položaju glede na –R ( v našem primeru R je –NH-Ph fragment, -NH-C=O fragment, -C=CH-Ph fragment) vezane na fenolni obroč ter da so te prav tako odgovorne za hormonsko aktivnost omenjenih spojin. V drugem delu doktorske disertacije smo potrdili hipotezi, da je hormonski učinek proučevanih spojin na zgoraj omenjene receptorje odvisen od hkratne prisotnosti drugih snovi in da imajo fenolni analogi BPA pleiotropni učinek. Poleg modulacije jedrnih receptorjev smo se osredotočili tudi na njihovo genotoksično in imunomodulatorno delovanje. Izvedli smo teste in vitro na LCL celični liniji za aminofenolne analoge BPA v smislu določanja vpliva izbranih spojin na produkcijo citokinov. Rezultati na LCL celični liniji kažejo, da imajo določeni aminofenolni analogi BPA imunomodulatorno delovanje pri koncentracijah, najdenih v človeški plazmi ter da na njega znatno vpliva metabolizem teh spojin. Dokazali smo, da lahko predvsem fenolni analogi BPA kot hormonski motilci glukokortikoidnega receptorja spremenijo imunski odziv izpostavljenih posameznikov. S pomočjo kvantno-kemijskih metod smo prvi predstavili mehanizem direktne poškodbe DNA toksičnega metabolita paracetamola (NAPQI). Paracetamol lahko uvrstimo med aminofenolne analoge BPA (tako kot diklofenak (DIC), 4-hidroksidiklofenak (4-HD) in piceatanol (PIC)). Ugotovili smo, da izkazuje aminofenolni analog BPA paracetamol preko svojega metabolita NAPQI nizko verjetnost genotoksičnosti, ki je možna le v primeru izčrpanih zalog glutationa v celici ter da so kinoniminske strukture manj reaktivne v primerjavi s kinonskimi. Predstavili smo podroben pregled vseh najpogosteje uporabljenih spojin, ki vsebujejo kinonsko in kinoniminsko strukturo ali vsebujejo to skupino njihovi metaboliti, ter posledice izpostavitve tem spojinam. Učinke mešanic izbranih spojin iz skupin onesnažil (propilparaben (PP), butilparaben (BP), dietilheksil ftalat (DEHP) in tetrametrin (TM)) in kozmetičnih sestavin (2-metilrezorcinol (2MR), avobenzon (AVB) in butil-hidroksianizol (BHA)) smo določili na celičnih linijah na enak način kot hormonske učinke posameznih spojin. Testiranja učinka mešanic smo se lotili na dva načina: proučevali smo mešanice izbranih spojin pri koncentracijah izpostavitve človeka (1 μM in 10 nM) ter pri koncentracijah, v katerih spojine izkazujejo 50 % spodbujevalni ali zaviralni učinek na proučevane receptorje (EC50 ali IC50). Glede na dobljene rezultate smo ugotovili, da testirane komponente mešanic interagirajo ena z drugo. Pokazali smo, da učinki mešanic niso zanemarljivi ter da jih je nujno potrebno vklopiti v oceno tveganja hormonskih motilcev na zdravje ljudi in njihovo okolje. Ti rezultati kažejo, da je potrebno natančno določiti strategijo za testiranje mešanic spojin, ki bo temeljila na ustreznih toksikoloških testih (že obstoječe in silico in in vitro metode, novi biološki testi ter Euromix modelna orodja za testiranje mešanic). In doctoral thesis we focused on the identification of new potential endocrine disrupting compounds, their pleiotropic effects and examined hormonal mixture effects thereof. As target group of tested compounds we chose phenolic analogues of bisphenol A (BPA) from the classes of drugs, pollutants and cosmetic ingredients. At first, we verify the hypothesis of the doctoral thesis whether the modulation of hormonal system depends on the chemical structure of the compounds. Among the phenolic analogues of BPA we identified recognizable structural elements responsible for the interaction with nuclear receptors. Using the freely accessible program package, Endocrine Disruptome, we predicted binding affinity to 12 different nuclear receptors for the selected compounds in silico. In particular, we focused on brominated flame retardants (TBB and TBPH), their metabolites (TBBA and TBMEPH) and aminophenol analogues of BPA (diclofenac (DIC), 4-hydroxydiclofenac (4- HD), paracetamol (PAR) and piceatannol (PIC)). Next, we tested them in vitro on two cell lines with luciferase reporting gene: on the MDA-kb2 cell line, which expresses the glucocorticoid (GR) and the androgen receptor (AR) and the GH3.TRE-Luc cell line expressing thyroid hormone receptors α and β (TRα and TRβ). For compounds which showed modulation of the glucocorticoid and androgen receptor, we additionaly performed an in vitro confirmatory test for binding. Among the first we have proved their different hormonal effects on estrogen (ER) and earlier mentioned receptors at concentrations, which represent real exposure levels on humans. We have found that the metabolism directly affects the modulation of the androgen, glucocorticoid and thyroid hormone receptors. For the modulation of the glucocorticoid receptor, the following functional groups are important: the free -COOH group and 2-(ethylhexyl)-oxycarbonyl group attached to the aromatic ring and the presence of three halogen atoms in the ortho position. The results showed that the antiandrogen activity depends on the number of carbonyl groups in the molecule and stilbenic scaffold without additional side chains between two benzene rings. We have found that the binding affinity for the androgen receptor depends on the presence Ph-N-C=O fragment, OH and NH-C=O groups, and the aromatic ring in their chemical structure. Also, the compounds with the brominated 3,5-substituent in the phenyl group possess antithyroid hormone effect. It has been observed, that the Ph-N-Ph fragment, that contains the Cl atom on one of the phenyl rings, is present in the compounds with mixed agonist/antagonist androgen activity. We have found that the endocrine disruptors of the androgen, glucocorticoid, thyroid hormone and estrogen receptors in their structures contain phenolic groups and also OH groups in para position with respect to the –R (in this case, R is –NH-Ph fragment, -NH-C=O fragment, - C=CH-Ph fragment) attached to a phenolic ring. In the second part of the doctoral thesis, we confirm the hypothesis, that the modulation of the above-mentioned receptors induced by the compounds depends on the presence of other compounds, and that the phenolic analogues of BPA have pleiotropic effect. In addition, we also focus on their genotoxic and immunomodulatory activities. We also performed in vitro assay on the LCL cell line in order to determine the immunomodulatory effect of the aminophenol analogues of BPA. Results obtained using the LCL cell line indicate that certain aminophenol analogues of BPA have immunomodulatory activity at concentrations found in human blood. We proved that the phenolic analogues of BPA as glucocorticoid receptor mimics of the natural ligand, may be associated with altered immune responsiveness. Making use of the available quantum-chemical methods, we first outline the mechanism of direct damage to the DNA by the toxic metabolite of the aminophenol analogue of BPA (NAPQI). Paracetamol can be classified as an aminophenol analogue of BPA (another are diclofenac (DIC), 4-hydroxydiclofenac (4-HD) and piceatannol (PIC)). We have found that the aminophenol analogue of BPA (PAR) possesses low genotoxicity, which is possible only in the case of the depleted glutathione, and that quinone imines are less reactive when compared to quinones. We presented a detailed overview of most commonly used compounds or their metabolites containing quinone and quinone imine structure as well as the consequences of exposure to these compounds. The effects of mixtures of compounds selected from the groups of pollutants (propylparaben (PP), butylparaben (BP), diethylhexyl phthalate (DEHP) and tetramethrin (TM)), and cosmetic ingredients (2-methylresorcinol (2MR), avobenzone (AVB) and butylated hydroxyanisole (BHA)) was determined in vitro in the same way as the hormonal effects of the individual compounds, explained above. We tested the mixture effects in two different ways: (i) selected compounds were mixed at concentrations to which humans are exposed (1 μM and 10 nM), (ii) at their EC50 or IC50 concentrations. Based on the obtained results, we found that the components of tested mixtures interact with each other. We pointed out, that the effects of mixtures are not negligible and that it is necessary to include them into the risk assessment of compounds. Also, it is necessary to specify a strategy for testing mixtures of compounds, which will be based on the relevant toxicological tests (already existing in silico and in vitro methods, new biological tests and Euromix modeling tools for testing mixtures).

Bisphenol A (BPA) is a toxic substance that is released into the environment mainly from paint, polymer, plastics and pharmaceutical industries. In this study, spinel Co3O4 consisting of Co2+/(Co3+)2O4 nanospheres with excellent specific surface properties were systematically synthesized via a simple hydrothermal approach preceded by calcination. The as-synthesized Co3O4 spinels were used for peroxymonosulfate (PMS) activation to reduce the bisphenol A (BPA). The effects of operating parameters including PMS dosage, initial pH, catalyst dosage, and co-existing ions were detected during the BPA degradation. It was observed that at neutral pH, the nano-Co3O4/PMS system effectively degrades the BPA (∼92%) with very low cobalt leaching and excellent recyclability. Control experiment analysis confirms the magnificent performance of the Co3O4/PMS system and the synergistic interaction between Co3O4 and PMS. Various characterization techniques were used to determine the thermal, textural and structural properties. A chemical quenching study confirmed that both hydroxyl radicals (•OH) and sulfate radicals (SO4•−) promotes BPA oxidation. The chloride (Cl-) ions and dihydrogen phosphate ions (H2PO4-) have little inhibition effect while adding humic acid (HA) and HCO3- inhibits the BPA oxidation. Separation techniques such as high-performance liquid chromatography (HPLC) coupled with mass spectroscopy (MS) were used to identify the oxidative by-products and the mineralization pathway of BPA reduction. First-order pseudo-kinetics were observed for BPA degradation. However, the power law model also fits nth-order kinetics models.

Bu tez çalışmasında, çevre ve sağlık üzerindeki toksik etkileri nedeniyle elektrokimyasal aktiviteye sahip olan Bisfenol A ve Bisfeno”l S’in elektroanalitik olarak, hızlı ve kolay bir şekilde algılanması amaçlanmıştır.Çalışmanın ilk aşamasında Bisfenol A ve Bisfenol S’in daha iyi algılanmasına imkan sağlayan çok duvarlı karbon nanotüpler-trietilentetramin (MWCNT-TETA) kullanılarak modifiye camsı karbon elektrot (CKE) hazırlanmıştır. Modifiye elekrot yalın CKE ile karşılaştırıldığında elektroanalitik performansının arttığı görülmektedir. Bisfenol A ve Bisfenol S’in elektrokimyasal özellikleri döngülü voltametri ve kare dalga voltametrisi ile irdelenmiştir. Bisfenol A ve Bisfenol S’in tekli tayinleri için doğrusal çalışma aralıkları sırasıyla 1,0-40 μM ve 1,0-120 μM gözlenebilme limitleri 0,26 ve 0,72 μM, eşzamanlı tayinlerinde ise her iki analit için de doğrusal aralıkları 1,0-30 μM arasında, gözlenebilme limit değerleri 0,35 ve 0,56 μM bulunmuştur. Yöntemin tekrarlanabilirliği ve stabilitesi araştırılmıştır. Son olarak hazırlanan sensör gıda örneklerinde BPA ve BPS’in eş zamanlı tayininde kullanılmıştır.Geliştirilen yöntem ile Bisfenol A ve Bisfenol S’in eş zamanlı olarak elektrokimyasal irdelenmesi hızlı, kolay ve güvenilir bir şekilde gerçekleştirilebilmektedir. In this thesis, it is aimed to detect Bisphenol A and Bisphenol S, which have electrochemical activity due to their toxic effects on the environment and health, electroanalytical, quickly and easily.In the first stage of the study, a modified glassy carbon electrode (GCE) was prepared using multi-walled carbon nanotubes-triethylenetetramine (MWCNT-TETA), which allows better detection of Bisphenol A and Bisphenol S. The electroanalytical performance of the modified electrode is improved when compared to the plain GCE. The electrochemical delebration of Bisphenol A and Bisphenol S were investigated by cyclic voltammetry (CV) and square wave voltammetry (SWV). Linear working ranges for single determinations of Bisphenol A and Bisphenol S are 1.0-40 μM and 1.0-120 μM, detection limits degree 0.26 and 0.72 μM, for simultaneous determinations linear ranges of 1.0-30 μM for both analytes, detection limits were found to be 0.35 and 0.56 μM, respectively. The reproducibility and stability of the method were investigated. Finally, the prepared sensor was used for the simultaneous determination of BPA and BPS in food samples.With the developed method, simultaneous electrochemical detection of Bisphenol A and Bisphenol S can be performed quickly, easily and reliably.

Cilj ovog rada bio je ispitati fitotoksičnost i oksidacijski stres kao mehanizam toksičnosti bisfenola A (BPA) u korjenčićima običnog luka (Allium cepa L.). Korjenčići lukovica luka (n = 10) bili su izloženi ili destiliranoj vodi (negativna kontrola) ili otopinama BPA (koncentracijski raspon 1-50 mg/l) kroz tri dana. Nakon trodnevne izloženosti, izvagana je masa svježeg tkiva korjenčića svake od lukovica te je pripremljen homogenat korjenčića luka u 5 % TCA (100 mg tkiva u 0,5 ml 5 % TCA). U supernatantu tako pripremljenog homogenata određeni su parametri oksidacijskog stresa: sadržaj reduciranog glutationa (GSH) i malondialdehida (MDA), a u talogu homogenata određen je sadržaj proteinskih karbonila. Sadržaj GSH određen je metodom prema Ellmanu (1959), MDA tiobarbituratnim testom, a proteinski karbonili metodom prema Levinu i sur. (1990), derivatizacijom karbonilne skupine proteina uz DNPH. Dobiveni rezultati statistički su obrađeni studentovim t-testom (Excell, MS Office). Masa svježeg tkiva korjenčića luka tretiranih otopinama BPA (1-50 mg/l) bila je niža u odnosu na negativnu kontrolu te je značajno smanjenje mase zabilježeno već nakon izloženosti BPA u koncentraciji 5 mg/l. Pad mase svježega tkiva s povećanjem koncentracije BPA ukazuje na fitotoksično djelovanje BPA na korjenčiće običnog luka. Sadržaj GSH bio je značajno niži u odnosu na kontrolu nakon izloženosti BPA u koncentraciji 1 mg/l. Značajan porast u odnosu na negativnu kontrolu sadržaja proteinskih karbonila zabilježen je nakon izloženosti BPA u koncentraciji 10 mg/l, a MDA nakon izloženosti 25 mg/l. Pad razine antioksidansa (GSH) i porast razine parametara oksidacijskog oštećenja lipida i proteina (MDA i proteinski karbonili) upućuju na to da je BPA izazvao oksidacijski stres u modelnom organizmu. Dobiveni rezultati pokazuju da BPA uzrokuje oksidacijsko oštećenje staničnih struktura te se može zaključiti da je oksidacijski stres jedan od mehanizama toksičnog učinka BPA. The aim of this study was to explore phytotoxicity and oxidative stress as a mechanism of bisphenol A (BPA) toxicity in onion roots (Allium cepa L.). Roots of onion bulbs (n = 10) were exposed to either distilled water (negative control) or BPA solutions (concentration range 1-50 mg/l) for three days. After a three-day exposure, the fresh root weight of each bulb was weighed, and in 5% TCA a homogenate of onion roots was prepared (100 mg of tissue in 0.5 ml of 5% TCA). In the supernatant of prepared homogenate oxidative stress parameters: the content of reduced glutathione (GSH) and malondialdehyde (MDA) was determined, and in the homogenate’s precipitate the content of protein carbonyls was assessed. The GSH content was determined based on the Ellman method (1959), MDA by using the thiobarbiturate test and the content of protein carbonyls was determined according to Levin et al. (1990), by derivatization of the proteins’ carbonyl group with DNPH. The obtained results were statistically analysed by student’s t-test (Excel, MS Office).The fresh tissue weight of onion roots treated with BPA (in concentration range 1-50 mg/l) was lower compared to the negative control, and a significant decrease in roots fresh weight was recorded already after exposure to BPA in concentration of 5 mg/l. The decrease in fresh tissue weight with increasing BPA concentration indicates that BPA had phytotoxic effect on onion roots. The exposure to BPA in concentration of 1 mg/l significantly decreased the content of GSH compared to the control. A significant increase compared to the negative control in the content of protein carbonyls was recorded after exposure to BPA in concentration of 10 mg/l, and MDA after exposure to BPA in concentration of 25 mg/l. A decrease in the level of antioxidants (GSH) and an increase in the level of lipid and protein oxidative damage parameters (MDA and protein carbonyls) indicate that BPA caused oxidative stress in the model organism. The obtained results imply that BPA caused oxidative damage to cellular structures, thus can be concluded that oxidative stress is one of the mechanisms of BPA toxicity.

[ES] El plástico es un material omnipresente, cuyo uso se ha incrementado con la pandemia de la COVID-19, con nefastas consecuencias para el medio ambiente. Además, una cantidad no despreciable de materiales plásticos que no son reciclados se acumula en el medio ambiente, donde se descomponen, transformándose en microplásticos, fragmentos con tamaño máximo de 5 mm. Por otra parte, los microplásticos sufren procesos de lixiviación que provocan la liberación de componentes tóxicos, alcanzando la cadena trófica. Entre ellos, el bisfenol A (BPA), que se emplea como monómero en policarbonatos (PC), o como aditivo en otros plásticos, es especialmente preocupante debido a su capacidad para unirse a receptores de estrógenos, siendo un potencial disruptor endocrino. Se han estudiado temperatura, pH, matriz de muestra, tiempo, tipo de radiación, parámetros que pueden influir en los procesos de lixiviación con microplásticos. Las condiciones que más se asemejan a los procesos naturales se han aplicado también a plásticos de PVC y PET. El análisis de BPA se ha realizado mediante cromatografía líquida de alta resolución con detección fluorescente. Además, en el caso de los lixiviados realizados en fase acuosa, ha sido necesario realizar un proceso de extracción en fase sólida (SPE), previo a su análisis, [EN] Plastic is an omnipresent material, whose use has been increased because of COVID19 crisis, with harmful consequences for the environment. Moreover, a considerable amount of non-recycled plastic accumulates in the environment, where they are decomposed and transformed into microplastics, fragments with a maximum size of 5 mm. On the other hand, microplastics undergo leaching processes, which cause the release of toxic components to the environments, which can enter in the food chain. Among them, bisphenol A (BPA), which is used as a monomer in polycarbonates (PC) or as an additive in other plastics, is particularly harmful due to its ability to bind to estrogen receptors, being a potential endocrine disruptor. Physicochemical parameters (temperature, pH, sample matrix, etc.) that can influence the leaching processes have been studied with PC microplastics. Finally, those conditions that resemble natural processes have been applied to PVC and PET ones. BPA analysis has been performed by high performance liquid chromatography with fluorescence detection. In addition, in the case of leachates made in aqueous phase, it has been necessary to carry out a solid phase extraction (SPE) process prior to analysis.

V zadnjih letih smo prišli do spoznanja, da smo izpostavljeni motilcem endokrinega sistema, ki se lahko zaradi svoje podobnosti v telesu prisotnim hormonom, vežejo na endokrine receptorje. Tako lahko delujejo bodisi kot agonisti ali kot antagonisti danih receptorjev. Eden izmed njih je bisfenol A, sintetična spojina, ki se uporablja v proizvodnji polikarbonatne plastike, epoksi smol in termalnega papirja. V človeško telo najpogosteje prehaja preko hrane ali plastičnih posod za hrano, vode, preko stika s termalnim papirjem, prisoten pa je tudi v zraku, predvsem kot posledica sežiganja plastike. Zaradi povečane prevalence različnih z endokrinim sistemom povezanih bolezni in ugotovljene povezave z bisfenolom A, se je njegova uporaba opazno zmanjšala. Zamenjali so ga njegovi analogi, ki so slabo raziskani. Namen našega dela je bil ugotoviti smotrnost zamenjave bisfenola A z njegovimi analogi z vidika učinka na estrogene receptorje. Testirali smo 13 analogov, preverili njihovo citotoksičnost z resazurinom in agonistični ter antagonistični učinek na estrogene receptorje α, ki smo ga izvedli na celicah hERα-HeLa-9903 z luciferaznim testom. Sledili smo smernicam OECD 455 in za vrednotenje agonističnega oziroma antagonističnega učinka izračunali parametre IC30, IC50, PC10, PC50, EC50 in RPCMAX. Citotoksični so bili bisfenoli C, G, PH pri koncentraciji 25 µM, zato teh koncentraciji nismo testirali za agonistični in antagonistični učinek. Kot agonisti estrogenskega receptorja α so se izkazali bisfenoli G, AP, B, E, C, S, dinitrobisfenol A, tetraklorobisfenol A in DD70. Kot antagonisti so se izkazali bisfenoli G, PH, dinitrobisfenol A, TGSH, BPSIP in DD70. Prišli smo do zaključka, da sta za vezavo v receptor ključni fenolni OH skupini na določeni razdalji in da estradiol najbolje oponašajo analogi, ki imajo substitucijo na C atomu med obema fenolnima obročema. Pri določanju antagonističnega učinka smo imeli več težav, odstopanja med meritvami so bila večja in posledično je bilo tudi več neskladij z že objavljeno literaturo, ki je je bilo na voljo manj, saj gre za slabše raziskan vidik vezave na estrogene receptorje. Za boljše razumevanje vpliva analogov bisfenola A na estrogenske receptorje bi bile potrebne nadaljnje preiskave. Vrednotili smo le aktivnost na estrogenske receptorje α, za bolj celovito sliko o varnosti analogov bisfenola A, bi bilo potrebno preveriti tudi učinke analogov na ostale estrogenske receptorje. In recent years, we have become aware of our exposure to endocrine disrupting chemicals (EDCs) that can bind to specific receptors because of their similarity to endogenous hormones. They can act as either agonists or antagonists of said receptors. One of the endocrine disrupting chemicals is bisphenol A, a synthetic chemical used in polycarbonate plastic, epoxy resins, and thermal paper. Exposure to bisphenol A occurs primarily through food, plastic food containers, water, thermal paper, and the air as plastic is burnt. Due to the increased incidence of various endocrine system disorders and their association with bisphenol A, it is being used less and less. It is replaced by its analogues, which are poorly studied. The aim of our work was to investigate the usefulness of replacing bisphenol A with its analogues in terms of its effect on estrogen receptors. We tested 13 analogues for cytotoxicity with resazurin, for agonistic and antagonistic effect on estrogen receptors α, which was performed on hERα-HeLa-9903 cells using the luciferase enzyme assay. We followed the OECD 455 protocol and calculated IC30, PC10, PC50, EC50, and RPCMAX to determine their agonistic or antagonistic effects. Bisphenols C, G, PH showed cytotoxicity at concentrations of 25 µM, so they were excluded from further evaluation. We concluded that bisphenols G, AP, B, E, C, S, dinitrobisphenol A, tetrachlorobisphenol A and DD70 showed agonistic effects on estrogen receptor α. On the other hand, bisphenols G, PH, dinitrobisphenol A, TGSH, BPSIP, and DD70 showed antagonistic effects. We had no major problems with the evaluation of agonistic effects, and our results were comparable to those previously published. We concluded that the phenolic OH groups with the correct spacing are crucial for binding to the receptor and that the analogues with a substitution at the C atom of the bridge best mimic estradiol. However, the evaluation of antagonistic effects was more problematic, and the variation between our results was larger, so our results were not necessarily in agreement with those of other research groups. We propose to investigate the effects of bisphenol A analogues on estrogen receptor α further to understand it better. We only studied the effects on estrogen receptors α for more accurate results, other estrogen receptors should also be studied.