Določitev aktivnega in pasivnega transporta parabenov skozi biološke bariere in vitro

Rigler, Ida (author), Žakelj, Simon (mentor)

Parabeni, estri para-hidroksibenzojske kisline, so eni izmed najpogosteje uporabljenih konzervansov v kozmetični, farmacevtski in prehranski industriji. Z njimi prihajamo v vsakdanjem življenju v stik po različnih poteh. Ne glede na pot izpostavitve se hitro absorbirajo in metabolizirajo. Metabolizem poteka z nespecifičnimi esterazami, pri čemer nastajata para-hidroksibenzojska kislina in pripadajoči alkohol. V sklopu diplomskega dela smo želeli natančneje preučiti mehanizem prehoda parabenov skozi biološke bariere. Zanimalo nas je predvsem, ali je v prehod parabenov skozi biološke bariere vpleten aktivni transport v eliminatorni smeri, ki bi lahko pojasnil nižjo permeabilnost parabenov. Poskušali smo tudi ugotoviti, kje v membrani prihaja do njihovega metabolizma v para-hidroksibenzojsko kislino. V ta namen smo uporabili Caco-2 celice in PAMPA (angl. Parallel Artificial Membrane Permeability Assay), kot biološki in nebiološki model za preučevanje permeabilnosti. Da bi določili, ali je aktivni transport v Caco-2 celicah prisoten, smo spremljali prehod snovi v absorptivni in eliminatorni smeri, dodatno pa smo aktivno komponento difuzije ovrednotili z dodatkom inhibitorja P-glikoproteina in prenašalca BCRP (angl. Breast Cancer Resistence Protein) na apikalno stran Caco-2 epitelija in vitro. Analizo pridobljenih vzorcev smo izvedli s HPLC sistemom. Rezultati poskusov so pokazali, da parabeni biološke bariere prehajajo predvsem s pasivno difuzijo. Aktivni transport torej ni mehanizem, ki zmanjšuje njihovo sistemsko absorpcijo. S preučevanjem metabolizma smo odkrili, da para-hidroksibenzojska kislina s hidrolizo parabenov nastaja v največji meri med samim prehodom membrane v notranjosti epitelijskih celic, poleg tega pa intenzivneje kot na bazolateralni nastaja na apikalni strani membrane. Metabolizem je torej v primeru parabenov poglavitni mehanizem, ki zmanjšuje njihovo biološko uporabnost, saj je sama para-hidroksibenzojska kislina zelo slabo permeabilna. Možna razlaga za večjo izpostavljenost parabenom ob peroralni aplikacijo kot ob dermalni aplikaciji bi lahko temeljila tudi na manjši aktivnosti esteraz v koži.

• Language: Slovenian
• Keywords: Parabeni, permeabilnost, biološke bariere, Caco-2, PAMPA
• Work type: Bachelor thesis/paper
• Organization: FFA - Faculty of Pharmacy
• Year: 2022
• PID: 20.500.12556/RUL-138901
• Publication date in RUL: 25.08.2022

Secondary language

• Language: English
• Title: Determination of active and passive transport of parabens through biological barrieres in vitro

Abstract

Parabens are esters of para-hydroxybenzoic acid and are one of the most commonly used preservatives in the cosmetic, pharmaceutical and food industries. We come into contact with them in different ways. Regardless of the route of exposure, they are rapidly absorbed and metabolized. Metabolism takes place with non-specific esterases, forming para-hydroxybenzoic acid and the associated alcohol. As part of our research, we wanted to study the mechanism of absorption of parabens through biological barriers. We were particularly interested if active transport in an eliminatory direction is involved, which could explain the lower permeability of parabens. We also tried to determine where in the membrane their metabolism to para-hydroxybenzoic acid occurs. For this purpose, we used Caco-2 cells and PAMPA as biological and non-biological models to study permeability. To determine if active transport of parabens was present on Caco-2 cells, we monitored the absorption of substances in an absorptive and eliminatory direction. The P-glycoprotein and BCRP inhibitor verapamil was also added to the apical side of the membrane. The obtained samples were analysed using an HPLC system. The results showed that parabens pass through biological barriers passively. Active transport is therefore not a mechanism that lessens their systemic absorption. By studying metabolism, we discovered that para-hydroxybenzoic acid is formed within the membrane as the parabens pass through it and is also more intensely produced on the apical side of the membrane. In the case of parabens, metabolism is therefore the primary mechanism that to some extent decreases their bioavailability, as para-hydroxybenzoic acid itself is very poorly permeable. The lower activity of esterases in the skin compared to the mucosa of the gastrointestinal tract could help explain why paraben exposure is greater when applied dermally.

• Keywords: Parabens, permeability, biological barriers, Caco-2, PAMPA