Mehanizam djelovanja fumonizina B1, bovericina i okratoksina A u bubrežnim stanicama

doctoral thesis

• Title (english): Mechanism of action of fumonisin B1, beauvericin and ochratoxin A in kidney cells
• Author: Maja Šegvić Klarić
• Mentor: Stjepan Pepeljnjak (mentor)
• Committee member: Jozsef Petrik (predsjednik povjerenstva)
• Committee member: Stjepan Pepeljnjak (član povjerenstva)
• Committee member: Maja Peraica (član povjerenstva); MBZ: 113106
• Granter: University of Zagreb; Faculty of Pharmacy and Biochemistry; (Department of microbiology); Zagreb
• Defense date and country: 2005, Croatia
• Scientific / art field, discipline and subdiscipline: BIOMEDICINE AND HEALTHCARE; Pharmacy; Pharmacy
• Universal decimal classification (UDC): 615 - Pharmacology. Therapeutics. Toxicology

Abstract

Fumonizin B1 (FB1), bovericin (BEA) i okratoksin A (OTA) su svjetski rašireni mikotoksini koji izazivaju čitav
niz patoloških promjena in vivo i in vitro, a čije je istodobno pojavljivanje u kukuruzu dokazano na području
Hrvatske. Stoga je u ovom radu ispitan mehanizam djelovanja FB1, BEA i OTA i njihove interakcije u PK 15
stanicama (epitel bubrega svinje), praćenjem preživljavanja stanica, integriteta plazmatske i mitohondrijske
membrane, razine lipidne peroksidacije i antioksidacijskog kapaciteta stanice te stupnja apoptoze i
genotoksičnog učinka niskih koncentracija mikotoksina (0,05 g/mL, 0,5 g/mL i 5 g/mL), tijekom 24 i 48
sati. Fumonizin B1, BEA i OTA su citotoksični za PK 15 stanice što se očituje smanjenjem vijabilnosti i
povećanjem katalitičke aktivnosti laktat dehidrogenaze i glutamat dehidrogenaze ovisno o dozi mikotoksina. Sva
tri mikotoksina u najvećoj koncentraciji značajno stimuliraju lipidnu peroksidaciju, a već pri nižim
koncentracijama smanjuju koncentraciju glutationa u PK 15 stanicama. Fumonizin B1, BEA i OTA uzrokuju
apoptozu PK 15 stanica pri čemu se značajnije morfološke apoptotičke promjene zamjećuju nakon 48-satnog
izlaganja. Okratoksin A ima jače djelovanje na aktivnost kaspaze-3 već nakon 24-satnog tretiranja u
koncentracijama od 0,5 i 5 g/mL, dok BEA i OTA uzrokuju značajnije povećanje aktivnosti ovog enzima
nakon duljeg izlaganja. FB1 i BEA su genotoksični za PK 15 stanice u koncentracijama od 0,5 i 5 g/mL, dok
OTA značajno povećava broj mikronukleusa već kod najmanje koncentracije. Sva tri mikotoksina imaju
klastogeni i aneugeni učinak pri čemu je klastogeno djelovanje OTA i BEA više izraženo. U koncentracijama
0,05 i 0,5 g/mL nakon 24- i 48-satnog izlaganja, kombinacije dva i sva tri mikotoksina mikotoksina pokazuju
uglavnom aditivane a manje sinergističke interakcije što se odražava na smanjenje vijabilnosti, narušavanje
integriteta stanične i mitohondrijske membrane, stimulaciju lipidne peroksidacije i smanjnje antioksidacijskog
kapaciteta te apoptozu i mutagenezu. Nakon 24-satnog izlaganja navedenim kombinacijama u koncentraciji 5
g/mL zapažen je aditivan, sinergistički i antagonistički učinak, a nakon 48-satnog tretmana u istoj koncentraciji
više je izražen antagonizam. Istodobni unos niskih koncentracija ovih mikotoksina putem hrane negativno se
odražava na antioksidacijski sustav te može pridonijeti imunosupresiji, nefrotoksičnosti i karcinogenezi u ljudi i
životinja tijekom dulje izloženosti.

Abstract (english)

Fumonisin B1 (FB1), beauvericin (BEA) and ochratoxin A are widespread mycotoxins, which cause a great
number of pathological changes in vivo and in vitro. Their co-occurrence was established in maize in Croatia.
The mechanism of action of FB1, BEA and OTA, as well as their interactions, were studied in the PK 15 cells
(porcine kidney epithelial cells). We examined the effects of low concentrations (0.05, 0.5 and 5 g/mL) of
individual and combined mycotoxins on cell viability, mitochondrial and plasma membrane integrity, lipid
peroxidation, antioxidative status, and apoptotic and genotoxic events in PK 15 cells. Decrease of cell viability
and increase of catalytic activity of LDH and GLDH show that FB1, BEA and OTA are cytotoxic to PK 15 cell
in dose dependent manner. Individual treatment of cells with 5 g/mL of FB1, BEA and OTA significantly
stimulated lipid peroxidation, while lower concentrations significantly reduced levels of glutathione. Significant
morphological apoptotic changes were observed after 48 hours of exposure to individual mycotoxins. Ochratoxin
A activated caspase-3 already after 24 hours of treatment with 0.5 and 5 g/mL, while BEA and OTA
significantly affected this enzyme after prolonged exposure. Fumonisn B1 and BEA are genotoxic to PK 15 cells
in concentrations of 0.5 and 5 g/mL, while OTA significantly increased the number of micronuclei already after
treatment with lowest concentrations. All of three mycotoxins mainly act as clastogens. Combined treatment
with two or all of three mycotoxins in concentrations of 0.05 and 0.5 g/mL during 24 and 48 hours, showed
mainly additive and less synergistic interactions on cell viability, mitochondrial and plasma membrane integrity,
stimulation of lipid peroxidation and decrease of antioxidative capacity, apoptotic and genotoxic events in PK 15
cells. After 24 hours of exposure to combined mycotoxins in concentration of 5 g/mL, additive, synergistic and
antagonistic effects were observed, while after 48 hours of exposure to same concentration we observed
antagonism. Simultaneous prolonged exposure to mycotoxins in low dietary concentrations can negatively affect
antioxidant status contributing the immunosuppression, nephrotoxicity and carcinogenicity in animals and
humans.

• Keywords: fumonizin B1; bovericin; okratoksin A; bubrežne stanice; citotoksičnost; antioksidacijski kapacitet; apoptoza; genotoksičnost
• Keywords (english): fumonisin B1; beauvericin; ochratoxin A; kidney cells; citotoxicity; antioxidative status; apoptosis; genotoxicity
• Language: croatian
• URN:NBN: urn:nbn:hr:163:505416
• Study programme: Title: Pharmacy and biochemistry; Study programme type: university; Study level: postgraduate; Academic / professional title: doktor znanosti (doktor znanosti)
• Type of resource: Text
• File origin: Born digital
• Access conditions: Open access
• Created on: 2017-11-23 14:44:35