Učinak citrinina na proteine Chk2 i FANCD2 u humanim staničnim linijama HepG2 i A549

master's thesis

• Title (english): Citrinin effect on Chk2 and FANCD2 proteins in human cell lines HepG2 and A549
• Author: Sonja Homar
• Mentor: Sanja Dabelić (mentor)
• Committee member: Sanja Dabelić (predsjednik povjerenstva)
• Committee member: Maja Šegvić Klarić (član povjerenstva)
• Committee member: Domagoj Kifer (član povjerenstva)
• Granter: University of Zagreb; Faculty of Pharmacy and Biochemistry; (Department of biochemistry and molecular biology); Zagreb
• Defense date and country: 2023-06-15, Croatia
• Scientific / art field, discipline and subdiscipline: BIOMEDICINE AND HEALTHCARE; Pharmacy; Medical Biochemistry

Abstract

Mikotoksini su produkti sekundarnog metabolizma filamentoznih gljivica. Citrinin, čest onečišćivač ljudske i stočne
hrane, poliketidni je mikotoksin koji pokazuje antibakterijska, antitumorska i neuroprotektivna svojstva, ali je
istovremeno jako nefrotoksičan i genotoksičan. Mehanizmi kojima ostvaruje svoju toksičnost su nedovoljno poznati, a
pretpostavlja se da je jedan od njih, kao i kod drugih mikotoksina, poticanje apoptoze te moduliranje različitih
signalnih puteva. U ovom radu istražen je učinak citrinina na ukupnu količinu i fosforilaciju dva proteina, Chk2 i
FANCD2, koji su ključni u mehanizmima dvolančanog popravka DNA i to na dvije stanične linije humanih tumorskih
stanica, HepG2 (hepatocelularni karcinom) i A549 (plućni adenokarcinom). Za ispitivanje je korišten s enzimom
povezani imunosorpcijski test (ELISA), CytoGlow™ koji se izvodi izravno na stanicama uzgajanima mikrotitarskim
pločicama. Dobiveni podaci analizirani su u statističkom programu GraphPad Prism. Utvrđeno je da citrinin može,
ukoliko je dovoljne koncentracije, uzrokovati povećanje ukupne količine proteina Chk2 i FANCD2 i u stanicama
HepG2 kao i u stanicama A549 te potaknuti njihovu fosforilaciju odnosno aktivaciju. Intenziteti uočenih promjena su
relativno blagi do osrednji, ovisni o primijenjenim koncentracijama i vrsti stanica. S obzirom da aktivaciju proteina
Chk2 i FANCD2 potiču dvolančani lomovi DNA, možemo pretpostaviti da je jedan od mehanizama kojima citrinin
ostvaruje svoju toksičnost upravo izazivanje takvih dvolančanih lomova DNA i moduliranje signalnih puteva u kojima
sudjeluju proteini Chk2 i FANCD2.

Abstract (english)

Mycotoxins are products of the secondary metabolism of filamentous fungi. Citrinin, a common food and animal feed
pollutant, is a polyketide mycotoxin, demonstrating antibacterial, antitumour and neuroprotective properties. It is also
highly nephrotoxic and genotoxic. Mechanisms of its toxicity are still insufficiently explored, but it is assumed that
one of those mechanisms, like in other mycotoxins, is apoptosis induction and modulation of various signaling
pathways. In this study, it was explored how citrinin impacts the total amount and phosphorylation of two proteins,
Chk2 and FANCD2, which are essential in double-strand DNA break repairs. The experiments were performed on two
human tumour cell culture lines, HepG2 (hepatocellular carcinoma) and A549 (lung adenocarcinoma), using
CytoGlow™ enzyme-linked immunosorbent assay (ELISA), directly on cells seeded in 96-well plates. The data was
analysed using the GraphPad Prism statistical software. It has been shown that citrinin can, if present in a high enough
dosage, cause the increase in total amounts of Chk2 and FANCD2 proteins, as well as induce their phosphorylation
and activation in both tested cell lines. The intensity of these changes is relatively mild to moderate, depending on the
concentration of citrinin which was applied and the type of cells. Because the activation of Chk2 and FANCD2 is
induced by double-stranded DNA breaks, we can assume that one of citrinin’s mechanisms of toxicity is precisely the
induction of double-stranded DNA breaks and the modulation of signaling pathways in which Chk2 and FANCD2
take part in.

• Keywords: citrinin; stanične linije; HepG2; A549; Chk2; FANCD2; mehanizam toksičnosti
• Keywords (english): citrinin; cell culture lines; HepG2; A549; Chk2; FANCD2; mechanism of toxicity
• Language: croatian
• URN:NBN: urn:nbn:hr:163:107316
• Study programme: Title: Medical biochemistry; Study programme type: university; Study level: integrated undergraduate and graduate; Academic / professional title: magistar/magistra medicinske biokemije i laboratorijske medicine (magistar/magistra medicinske biokemije i laboratorijske medicine)
• Type of resource: Text
• File origin: Born digital
• Access conditions: Open access; Embargo expiration date: 2023-12-15
• Created on: 2023-11-08 10:06:57