Sinteza bisfenol S sulfata ter vrednotenje encimske kinetike sulfatiranja bisfenola S in vitro

Jurić, Monika (avtor), Peterlin Mašič, Lucija (mentor), Trontelj, Jurij (komentor)

Bisfenol A ima dokazane neželene učinke na zdravje ljudi, zaradi česar so prepovedali njegovo uporabo pri izdelavi otroških stekleničk in termičnega papirja. Posledično se je s tem razširila uporaba njegovih analogov, med drugimi tudi bisfenola S. Bisfenol S se uporablja kot monomer pri izdelavi polikarbonatne plastike in epoksi smol, iz katerih so narejene plastenke, vsebniki za hrano, otroške stekleničke in igrače. Zaradi podobne strukture kot bisfenol A, ima tudi bisfenol S agonistično estrogeno delovanje in ga uvrščamo med kemične motilce endokrinega sistema, vendar so neželeni učinki v primerjavi z BPA slabše raziskani. BPS lahko vpliva na nastanek in razvoj različnih bolezni, povzroča tudi motnje v razvoju ploda. Zaradi toksičnih učinkov je pomembno, da se čim hitreje izloči iz telesa. Najbolj znana in raziskana presnovna pot BPS je glukuronidacija, ki jo katalizirajo encimi iz družine uridindifosfat glukuronoziltransferaz. Sulfatiranje bisfenola S, v prisotnosti sulfotransferaz poteka v manjši meri in je zaradi slabše raziskanosti glavna tema te magistrske naloge. Sulfatiranje bisfenola S smo izvedli in vitro, s citosolnimi frakcijami človeških jeter, ki za razliko od mikrosomov vsebujejo sulfotransferaze. Po določitvi pogojev začetne hitrosti reakcije sulfatiranja, smo le-to izvedli in vzorce analizirali z metodo LC-MS/MS. Z izdelavo in uporabo umeritvene krivulje smo dobili koncentracije nastalega BPS sulfata ter izračunali hitrost nastajanja produkta. S pomočjo Michaelis-Mentenove enačbe smo izračunali navidezne encimske parametre K_M (71,74 µmol/L) in v_max (0,8086 nmol/min/mg proteinov) ter intrinzični očistek (0,0113 mL/min/mg), ki smo ga ekstrapolirali na celoten organ (19,47 mL/min/kg). Za boljše razumevanje metabolizma bisfenola S, smo pridobljene parametre sulfatiranja primerjali s parametri glukuronidiranja, ki so že bili opisani v različnih študijah. Ugotovili smo, da imajo sulfotransferaze poleg uridindifosfat glukuronoziltransferaz zmerno visoko afiniteto do bisfenola S, vendar je hitrost njegovega sulfatiranja mnogo nižja od hitrosti glukuronidiranja. To pomeni, da v metabolizmu bisfenola S glukuronidacija prevlada, kar se sklada tudi s podatki iz ostalih objavljenih študij in vitro in in vivo.

• Jezik: Slovenski jezik
• Ključne besede: bisfenol S, kemični motilci endokrinega sistema, sulfatiranje, sulfotransferaze, encimska kinetika
• Vrsta gradiva: Magistrsko delo/naloga
• Organizacija: FFA - Fakulteta za farmacijo
• Leto izida: 2021
• PID: 20.500.12556/RUL-124666
• Datum objave v RUL: 06.02.2021

Sekundarni jezik

• Jezik: Angleški jezik
• Naslov: Synthesis of bisphenol S sulfate and evaluation of the kinetics of bisphenol S sulfation in vitro

Izvleček

Due to the proven adverse effects on human health, the authorities banned the use of bisphenol A and thus expanded the use of its analogues. One of them is also bisphenol S which is used as a monomer in the manufacture of polycarbonate plastics and epoxy resins from which bottles, food containers, baby bottles and toys are made. Due to its similar structure to bisphenol A, bisphenol S too exhibits endocrine activity and is classified as a chemical disruptor of the endocrine system, however it is less well studied compared to bisphenol A. It can affect the onset and development of various diseases, furthermore it also causes disorders in fetal development. As it endangers our health, it is important that it is eliminated from the body as rapidly as possible. The most known and researched metabolic pathway of bisphenol S is glucuronidation, which is catalyzed by enzymes from the uridine diphosphate glucuronosyltransferase family. Sulfation of bisphenol S in the presence of sulfotransferases takes place to a lesser extent and is due to poor research also the main topic of this master's thesis. Sulfation of bisphenol S was performed in vitro with cytosolic fractions of human liver, which, unlike microsomes, contain sulfotransferases. After determining the initial reaction rate conditions, bisphenol S sulfation was performed and samples were analyzed by LC-MS/MS method. The calibration curve method was used to obtain the concentrations of the formed bisphenol S sulphate and to calculate the rate of product formation. Using the Michaelis-Menten equation, the apparent enzyme parameters K_M (71,74 µmol/L) and v_max (0,8086 nmol/min /mg protein) were calculated. Intrinsic clearance (0,0113 mL/min /mg) was also calculated and extrapolated per whole organ (19,47 mL/min/kg). To better understand bisphenol S metabolism, the obtained sulfation parameters were finally compared with the glucuronidation parameters described in various studies. Our results show that while the sulfotransferases have a moderately high affinity towards bisphenol S, the reaction rate is relatively low, therefore the glucuronidation pathway prevails over sulfation in bisphenol S metabolism, which also corresponds with data from other in vitro and in vivo studies.

• Ključne besede: bisphenol S, endocrine disrupting chemicals, sulfation, sulfotransferases, enzyme kinetics