The combined effects of 2,3,7,8-tetrachlorodibenzo-<i>p</i>-dioxin and the phytoestrogen genistein on steroid hormone secretion, AhR and ERβ expression and the incidence of apoptosis in granulosa cells of medium porcine follicles

Abstract

Low doses of endocrine disrupting chemicals (EDCs) used in combination may act in a manner different from that of individual compounds. The objective of the study was to examine in vitro effects of low doses of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD; 100 pM) and genistein (500 nM) on: 1) progesterone (P4) and estradiol (E2) secretion (48 h); 2) dynamic changes in aryl hydrocarbon receptor (AhR) mRNA and protein expression (1, 3, 6, 24 and 48 h); 3) dynamic changes in estrogen receptor β (ERβ) mRNA and protein expression (1, 3, 6, 24 and 48 h); and 4) induction of apoptosis in porcine granulosa cells derived from medium follicles (3, 6 and 24 h). TCDD had no effect on P4 or E2 production, but potentiated the inhibitory effect of genistein on P4 production. In contrast to the individual treatments which did not produce any effects, TCDD and genistein administered together decreased ERβ and AhR protein expression in granulosa cells. Moreover, the inhibitory effect of TCDD on AhR mRNA expression was abolished by genistein. The treatments did not induce apoptosis in the cells. In summary, combined effects of low concentrations of TCDD and genistein on follicular function of pigs differed from that of individual compounds. The results presented in the current paper clearly indicate that effects exerted by low doses of EDCs applied in combination must be taken into consideration when studying potential risk effects of EDCs on biological processes.

Identification and characterization of long non-coding RNAs in porcine granulosa cells exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin

“…In the current study, we investigated the genome-wide lncRNA expression profile of porcine granulosa cells exposed to TCDD. The effects of TCDD on the porcine granulosa cell transcriptome have been previously examined, however the studies have been focused solely on the expression of genes (RNA-Seq [ 23 ]; real-time PCR [ 29 ]). In comparison to humans and rodents, information concerning the identification and expression of lncRNAs in pigs is limited [ 24 , 50 ].…”

“…To bring out the potential of TCDD to transduce intracellular signaling in the examined cells, the selected dose of TCDD moderately exceeded its environmentally relevant concentration. Moreover, 100 nmol/L of TCDD was found to be effective in porcine granulosa cells (steroidogenesis: [ 26 – 28 ]; gene expression: [ 23 , 29 ]). At the end of the culture, medium was removed, cells were washed twice with a phosphate-buffered saline (PBS; Sigma Aldrich) and designated for total RNA isolation.…”

Effect of dioxins in milk on 3D-cultured primary buffalo granulosa cells, a pilot study for a prospective RT-LAMP colour reaction for dioxin toxicity

“…This kind of different AHR gene expression under different TCDD treatments could be due to the differences between the AHR mRNA and protein levels. In porcine granulosa cell culture, TCDD decreased AHR protein expression after 1hr and 48 hr, but AHR mRNA expression was influenced only after 48 h (Piasecka-Srader et al 2016). This shows that TCDD induces rapid reduction of AHR protein prior to its significant effect on AHR mRNA expression in in vitro cell culture systems.…”

“…These observations indicate that the ER-beta could be another potential gene marker like AHR gene to identify the dioxins in milk. This similarity in the ER-beta and AHR expression could be due a crosstalk between AHR and estrogen receptors, particularly ER-beta rather than ER-alpha (Piasecka-Srader et al, 2016). It is well known that the granulosa cells of small follicles in most species including rat and human express ER-beta as a dominant isoform of estrogen receptors in their nuclei (Chu et al, 2004).…”

The effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on the transcriptome of aryl hydrocarbon receptor (AhR) knock-down porcine granulosa cells

“…In the present study, we were focused on identifying all possible pathways (molecules) involved in the mechanism of TCDD action in porcine granulosa cells and, therefore, the selected dose of TCDD (100 nM) moderately exceeded its environmentally relevant concentrations. The concentration of 100 nM of TCDD was previously reported to affect porcine granulosa cells steroidogenesis (Gregoraszczuk et al, 2000;Gregoraszczuk, 2002;Jablonska et al, 2014), gene expression (Piasecka-Srader et al, 2016;Sadowska et al, 2017;Nynca et al, 2019), lncRNA expression (Ruszkowska et al, 2018) and proteome (Orlowska et al, 2018) without affecting cell viability and morphology. Moreover, 100 nM was one of the two most frequently used TCDD concentrations in in vitro experiments performed on porcine ovarian cells.…”