2,3,7,8-Tetrachlorodibenzo-p-dioxin alters steroid secretion but does not affect cell viability and the incidence of apoptosis in porcine luteinised granulosa cells

Abstract

The compound 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), a by-product of human industrial activity, was found to affect ovarian steroidogenesis in animals, but the mechanism of its action is still unclear. The aims of the study were to examine the effect of TCDD on (1) progesterone (P 4 ) and oestradiol (E 2 ) production by granulosa cells isolated from medium (3-6 mm) and preovulatory (≥ 8 mm) porcine follicles, (2) the viability of the cells, and (3) the incidence of apoptosis. Porcine granulosa cells were cultured (48 h) with or without TCDD (100 pM, 100 nM). Steroid hormone concentrations in the medium were determined by radioimmunoassay. The viability of granulosa cells was tested spectrophotometrically (alamarBlue™ assay). Apoptosis was evaluated by flow cytometry using Annexin V and by TUNEL assay. The higher dose of TCDD (100 nM) significantly inhibited P 4 and stimulated E 2 production by luteinised granulosa cells isolated from medium follicles. The lower dose of TCDD (100 pM) significantly stimulated P 4 and inhibited E 2 secretion by the cells isolated from preovulatory follicles. None of the two TCDD doses affected cell viability or induced apoptosis in granulosa cells. In conclusion, TCDD directly affected steroid production by granulosa cells obtained from mature pigs, but the effect of TCDD was not due to its cytotoxicity.

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.…”

“…Since TCDD influences the production of estrogens and progesterone by porcine granulosa cells (Gregoraszczuk, 2002;Jablonska et al, 2011;Jablonska et al, 2014), it is of importance to identify its molecular targets in follicular cells. The results of our previous studies, performed on porcine granulosa cells, demonstrated, among others, that TCDD affected the expression of transcripts involved in the follicular atresia as well as cell proliferation and cell cycle regulation (Sadowska et al, 2017;Ruszkowska et al, 2018;Nynca et al, 2019).…”

Endocrine Disrupting Chemicals and Endometrial Cancer: An Overview of Recent Laboratory Evidence and Epidemiological Studies

“…Many of the congeners are proved to be highly toxic, but 2,3,7,8-tetrachlorodibenzo- p -dioxin (TCDD) is the most toxic congener, and probably the most toxic compound ever synthesized by man, being widespread and highly persistent in the environment, and accumulating in living organisms, especially in fat tissue. The dioxins are known to adversely affect the reproductive system in part due to their ability to alter hormone levels in many species [84,85]. …”

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

“…TCDD is widespread and highly persistent in the environment and accumulates in living organisms, especially in fat tissue. The dioxin is known to adversely affect reproduction in part due to its ability to alter hormone levels in many species, including pigs [ 7 , 8 ]. Previous studies indicate that most of TCDD effects are mediated through the aryl hydrocarbon receptor (AhR), a ligand-activated member of the bHLH-PAS family of transcription factors [ 9 ].…”

“…Therefore, the goal of this study was to establish the influence of low doses of TCDD and genistein used in combination on porcine granulosa cells in vitro . Since extensive dose dependent studies on porcine granulosa cells have been already performed individually for genistein [ 5 , 15 , 16 ] and TCDD [ 7 , 8 , 17 ], the current study was focused on the examination of the combined effects of the low but environmentally relevant doses of the two treatments. Thus, the specific objectives were to examine the effects of TCDD (100 pM) and/or genistein (500 nM) on: 1) progesterone (P 4 ) and estradiol (E 2 ) production; 2) dynamic changes in expression of AhR (mRNA and protein); 3) dynamic changes in expression of ERβ (mRNA and protein); and 4) induction of apoptosis in porcine granulosa cells derived from medium (3–6 mm) porcine follicles.…”