Vpliv bisfenola A na viabilnost in izražanje genov apoptoze in steroidogeneze v celicah granuloze iz foliklove tekočine in tumorskih celicah celične linije KGN v pogojih in vitro

Celar Šturm, Dominika (author), Virant Klun, Irma (mentor), Geršak, Ksenija (comentor)

Izvleček Uvod: Bisfenol A (BPA) je hormonski motilec, ki preko okoljske izpostavljenosti, predvsem sproščanja iz plastike, vstopa v človeško telo, kjer lahko zaradi svoje sposobnosti vezave na estrogenske receptorje deluje kot hormonski motilec in negativno vpliva na reproduktivno zdravje. V raziskavi smo preučevali vpliv BPA na »zdrave« celice granuloze iz foliklove tekočine, ključne gradnike jajčnikovih foliklov in tumorske celice granuloze iz celične linije KGN. Zanimal nas je učinek BPA na celično število, viabilnost in izražanje genov, povezanih z apoptozo in steroidogenezo, preverili pa smo tudi njegov vpliv na sproščanje estradiola in progesterona v rastno gojišče z analizo gojišča po zaključenem gojenju celic. Metode: Foliklovo tekočino smo po podpisanem informiranem pristanku pridobili od treh bolnic, udeleženih v postopek zunajtelesne oploditve zaradi moške neplodnosti. Celice granuloze smo iz foliklove tekočine izolirali z uporabo centrifugiranja na gradientu 50 % PureSperm (Nidacon, Švedska), nato pa jih nacepili in gojili v standardnih pogojih v rastnem gojišču DMEM-F12 z dodanim telečjim fetalnim serumom in antibiotiki na enak način kot tumorske celice granuloze KGN. V raziskavi smo pri dveh časovnih izpostavitvah (24 in 72 h) uporabili tri različne koncentracije BPA, dve koncentraciji (0,001 in 0,1 µM), primerljivi z izmerjenimi koncentracijami v različnih bioloških vzorcih človeka, ter toksikološko koncentracijo (100 µM), s katero smo želeli preveriti morebitne citotoksične učinke. Število in viabilnost celic smo izmerili s pomočjo avtomatskega števca celic in barvila tripansko modrilo, rast celic KGN pa smo spremljali tudi s fazno kontrastno mikroskopijo v časovnih presledkih. Izražanje genov, povezanih z apoptozo in steroidogenezo, smo analizirali z metodo verižne reakcije s polimerazo v realnem času (RT-qPCR) s predpripravljenimi ploščami za sočasno, hkratno analizo 96 genov, udeleženih v apoptozi (TaqManTM Array, Human Cellular Apoptosis Pathway, Fast 96-well, Applied Biosystems/Thermo Fisher Scientific, ZDA), in 96 genov, udeleženih v steroidogenezi (TaqManTM Array, Human Estrogens, Applied Biosystems/Thermo Fisher Scientific, ZDA) v primerjavi s kontrolo, nadalje pa smo za izbrane gene analizirali izraženost pripadajočih proteinov z metodo prenosa Western. Koncentracijo estradiola in progesterona v gojišču smo analizirali z elektrokemiluminiscenčnim imunskim testom (ECLIA). Rezultate smo statistično vrednotili z metodama enosmerna in dvosmerna ANOVA. Rezultati: BPA po 24-urni izpostavitvi celic ni vplival na noben izbrani parameter celic in pri nobenem celičnem modelu. Po 72 h je BPA pri najvišji koncentraciji (100 µM) tako pri celicah granuloze iz foliklove tekočine kot pri tumorskih celicah KGN povzročil statistično značilno zmanjšano viabilnost, pri nižjih koncentracijah pa nismo zaznali statistično značilnega vpliva v primerjavi s kontrolo. BPA je pri vseh koncentracijah statistično značilno (p < 0,05) vplival na izražanje genov, povezanih tako z apoptozo kot steroidogenezo, vendar so se ti geni večinoma razlikovali med celicami granuloze iz foliklove tekočine in tumorskimi celicami KGN. Med geni, povezanimi z apoptozo, smo pri koncentraciji 0,001 µM BPA v celicah granuloze iz foliklove tekočine zaznali povečano izražanje gena PPID in zmanjšano izražanje genov CASP3 in RPS6KA3, pri koncentraciji 0,1 µM BPA pa povečano izraženost genov BID, PPID in IKBKG ter zmanjšanje izraženosti gena CASP3 v primerjavi s kontrolo. Najvišja koncentracija BPA (100 µM) je povzročila statistično značilno povečano izražanje genov BID, PPID, IKBKG in težnjo k povečani izraženosti gena CASP3. Pri celicah KGN smo vpliv BPA na izraženost genov, povezanih z apoptozo, zaznali le pri najvišji koncentraciji 100 µM, kjer smo ugotovili povečano izražanje genov RIPK1, RPS6KA5 in težnjo k povečani izraženosti genov FADD in BBC3. Med geni, povezanimi s steroidogenezo, smo v celicah granuloze iz foliklove tekočine pri izpostavljenosti 0,001 µM BPA zaznali značilno povečano izražanje gena NR6A1 in zmanjšano izražanje genov UGT2B15 in TRIM25, pri koncentraciji 0,1 µM BPA pa povečano izraženost genov AR in HSD3B1 ter zmanjšano izraženost gena TRIM25. Pri najvišji koncentraciji BPA (100 µM) smo ugotovili statistično značilno povečano izražanje genov AR, CYP1B1 in GPR30 in sočasno zmanjšano izražanje genov FOXO1 in UGT2B15 v primerjavi s kontrolo. Pri tumorskih celicah KGN smo vpliv BPA na izražanje genov, povezanih s steroidogenezo, zaznali le pri srednji in najvišji koncentraciji BPA, kjer smo ugotovili zmanjšano izražanje gena CYP11A1 pri 0,1 µM BPA in povečano izraženost genov ESRRA, FOXO1, OVGP1 in GPR30 pri 100 µM BPA. Tudi pri merjenju koncentracije estradiola in progesterona, sproščenega v gojišče, smo vpliv zaznali le pri najvišji koncentraciji BPA (100 µM). Toksikološka koncentracija BPA je povzročila statistično značilno nižjo koncentracijo estradiola in progesterona v rastnem gojišču pri celicah granuloze iz foliklove tekočine, kar smo za progesteron potrdili tudi po normalizaciji na 1000 živih celic, za estradiol pa ne. Zaključek: V raziskavi je BPA izkazal citotoksični učinek na število in viabilnost celic granuloze iz foliklove tekočine in negativno vplival na sproščanje estradiola in progesterona v gojišče le pri najvišji koncentraciji 100 µM, učinek na izraženost analiziranih genov, povezanih z apoptozo in steroidogenezo, pa je bil opazen tudi pri nižjih koncentracijah BPA. Spremembe v izražanju genov so se razlikovale glede na koncentracijo BPA in celični model. Med geni, povezanimi z apoptozo, bi izpostavili povečano izražanje pro-apoptotskega gena BID v celicah granuloze iz foliklove tekočine, izpostavljenih 0,1 µM ali 100 µM BPA. Med geni, povezanimi s steroidogenezo, pa je izpostavljenost celic BPA predvsem povečevala izraženost genov za hormonske receptorje: receptor za androgene (AR) pri celicah granuloze iz foliklove tekočine, z estrogenom povezan receptor (ESSRA) pri tumorskih celicah KGN in za z G-proteinom povezan receptor za estrogen (GPR30), tako pri celicah granuloze iz foliklove tekočine kot tudi tumorskih celicah KGN. Negativne učinke BPA smo pri vseh meritvah opazili šele po daljši izpostavljenosti celic, tj. 72 ur, kar kaže na to, da se učinki BPA večajo s časom izpostavljenosti. Celice granuloze iz foliklove tekočine so se izkazale za bolj občutljive kot tumorske celice KGN, saj smo spremembe v izražanju genov, povezanih z apoptozo in steroidogenezo, zaznali pri vseh koncentracijah BPA, pri tumorskih celicah KGN pa razen enega gena samo pri najvišji koncentraciji BPA 100 µM.

• Language: Slovenian
• Keywords: Bisfenol A, BPA, Jajčnik, Folikel, Celice granuloze, Celice KGN, Steroidogeneza, Apoptoza, Hormonski motilci, Estradiol, Progesteron
• Work type: Doctoral dissertation
• Organization: MF - Faculty of Medicine
• Year: 2026
• PID: 20.500.12556/RUL-180891
• Publication date in RUL: 19.03.2026

Secondary language

• Language: English
• Title: The effect of bisphenol A on the viability and expression of apoptotic and steroidogenesis-related genes in granulosa cells from the folicular fluid and tumour cells of the KGN cell line under in vitro conditions

Abstract

Abstract Introduction: Bisphenol A (BPA) is an endocrine disruptor that enters the human body through environmental exposure, primarily via leaching from plastic materials. Due to its ability to bind to estrogen receptors, BPA can act as a hormonal disruptor and negatively affect reproductive health. In this study, we investigated the effect of BPA on "healthy" granulosa cells isolated from human follicular fluid, which are key components of ovarian follicles, as well as on tumor-derived granulosa cells from the KGN cell line.. We assessed the effects of BPA on cell number, viability and gene expression related to apoptosis and steroidogenesis, and also examined its impact on the secretion of estradiol and progesterone into the culture medium by analyzing the spent media after cell cultivation. Methods: Follicular fluid was obtained from three patients undergoing in vitro fertilization procedure due to male-factor infertility, after signing informed consent. Granulosa cells were isolated using 50% PureSperm (Nidacon, Sweden) density gradient centrifugation, then seeded and cultured under standard conditions in DMEM-F12 growth medium supplemented with fetal bovine serum and antibiotics, using the same protocol for the KGN tumor cell line. We used three different concentrations of BPA across two exposure time points (24 and 72 h): two concentrations (0.001 and 0.1 µM), comparable to those measured in various human biological samples, and one toxicological concentration (100 µM) to evaluate potential cytotoxic effects Cell number and viability were measured using an automatic cell counter and trypan blue staining, and KGN cell growth was also monitored by phase-contrast microscopy at various time intervals. Gene expression related to apoptosis and steroidogenesis was analyzed using real-time quantitative polymerase chain reaction (RT-qPCR) with pre-configured 96-well arrays for simultaneous analysis of 96 apoptosis-related genes (TaqManTM Array, Human Cellular Apoptosis Pathway, Fast 96-well, Applied Biosystems/Thermo Fisher Scientific,ZDA) and 96 steroidogenesis-related genes (TaqManTM Array, Human Estrogens, Applied Biosystems/Thermo Fisher Scientific, ZDA), compared to control. For selected genes, belonging protein expression was further analyzed by Western blot. The concentrations of estradiol and progesterone in the spent culture media were measured using electrochemiluminescence immunoassay (ECLIA). The results were statistically analyzed using one-way and two-way ANOVA. Results: BPA showed no effect on any selected cellular parameters after 24-hour exposure in either cell model. After 72 h, the highest BPA concentration (100 µM) led to a statistically significant reduction in viability in both follicular fluid-derived granulosa cells and KGN tumor cells, while lower concentrations of BPA did not show any significant differences compared to control. BPA significantly (p < 0.05) affected the expression of genes related to apoptosis and steroidogenesis at all concentrations, though the specific genes which were involved differed between granulosa cells from follicular fluid and tumor KGN cells. Among apoptosis-related genes, 0.001 µM BPA resulted in increased expression of PPID and decreased expression of RPS6KA3 genes and in tendency to decreased expression of gene CASP3 in follicular fluid granulosa cells. At 0.1 µM BPA, genes BID, PPID, and IKBKG were upregulated, while gene CASP3 was downregulated compared to control. The highest BPA concentration (100 µM) significantly upregulated genes BID, PPID, IKBKG, and showed a tendency to increased CASP3 expression. In KGN cells, BPA-induced changes in apoptosis gene expression were observed only at 100 µM BPA, including increased expression of genes RIPK1 and RPS6KA5, and a trend toward increased FADD and BBC3 expression. Among steroidogenesis genes, 0.001 µM BPA in follicular fluid granulosa cells led to increased expression of NR6A1 and decreased expression of UGT2B15 and TRIM25 genes. At 0.1 µM BPA, genes AR and HSD3B1 were upregulated, while TRIM25 was downregulated. The highest BPA concentration 100 µM significantly increased AR, CYP1B1, and GPR30 expression, and decreased FOXO1 and UGT2B15 expression compared to control. In KGN tumor cells, BPA affected steroidogenesis-related gene expression only at medium and high concentrations: gene CYP11A1 was downregulated at 0.1 µM BPA, while genes ESRRA, FOXO1, OVGP1, and GPR30 were upregulated at 100 µM BPA. A measurable effect on estradiol and progesterone secretion into spent culture medium was detected in follicular fluid granulosa cells only at the highest BPA concentration 100 µM. This toxicological concentration of BPA led to a statistically significant decrease in estradiol and progesterone concentrations in the spent culture media of follicular fluid granulosa cells; this decrease remained statistically significant for progesterone even after normalization to 1000 live cells, but not for estradiol. Conclusion: In this study, BPA exhibited cytotoxic effects on the viability of follicular fluid-derived granulosa cells and reduced estradiol and progesterone secretion into the culture medium only at the highest concentration of 100 µM. However, changes in gene expression related to apoptosis and steroidogenesis were observed even at lower BPA concentrations, 0.001 M and 0.M. Gene expression changes varied depending on BPA concentrations and cell model. Among apoptosis-related genes, increased expression of the pro-apoptotic gene BID in follicular fluid granulosa cells was observed after exposure to 0.1 µM or 100 µM BPA. Among steroidogenesis-related genes, BPA exposure primarily increased expression of hormone receptor genes: androgen receptor (AR) in follicular fluid granulosa cells, estrogen-related receptor (ESSRA) in KGN tumor cells, and the G protein-coupled estrogen receptor (GPR30) in both granulosa cell types. Negative BPA effects were observed only after longer exposure (72 h), indicating a time-dependent cell response. Follicular fluid granulosa cells appeared to be more sensitive than KGN tumor cells, as gene expression changes in apoptosis and steroidogenesis-related genes were detected at all BPA concentrations, whereas in KGN tumor cells, changes (except for one gene) were limited to the highest BPA concentration (100 µM) only.

• Keywords: Bisphenol A, BPA, Ovary, Follicle, Granulosa cells, KGN cells, Steroidogenesis, Apoptosis, Endocrine disruptors, Estradiol, Progesterone