Di-(2-ethylhexyl) phthalate (DEHP) inhibits steroidogenesis and induces mitochondria-ROS mediated apoptosis in rat ovarian granulosa cells

• Published in: Toxicology research (Cambridge) Vol. 8; no. 3; pp. 381 - 394
• Main Authors: Tripathi, Anima, Pandey, Vivek, Sahu, Alakh N, Singh, Alok, Dubey, Pawan K
• Format: Journal Article
• Language: English
• Published: England Royal Society of Chemistry 01-05-2019
• Subjects: 17β-Estradiol; Acetylcysteine; Apoptosis; Bcl-2 protein; Caspase; Caspase-3; Chemistry; Conditioning; Cytochrome; Cytochrome c; Cytochromes; Galactosidase; Gene expression; Genes; Granulosa cells; Hormones; Membrane potential; Mitochondria; Oocytes; Oxidative stress; Progesterone; Reactive oxygen species; Senescence; Sex hormones; Steroid hormones; Steroidogenesis; Steroids; β-Galactosidase
• ISSN: 2045-452X; 2045-4538
• DOI: 10.1039/c8tx00263k

Increased oxidative stress (OS) due to ubiquitous exposure to di-(2-ethylhexyl) phthalate (DEHP) can affect the quality of oocytes by inducing apoptosis and hampering granulosa cell mediated steroidogenesis. This study was carried out to investigate whether DEHP induced OS affects steroidogenesis and induces apoptosis in rat ovarian granulosa cells. OS was induced by exposing granulosa cells to various concentrations of DEHP (0.0, 100, 200, 400 and 800 μM) for 72 h
in vitro
. Intracellular reactive oxygen species (ROS), oxidative stress (OS), mitochondrial membrane potential, cellular senescence, apoptosis, steroid hormones (estradiol & progesterone) and gene expression were analyzed. The results showed that an effective dose of DEHP (400 μg) significantly increased OS by elevating the ROS level, mitochondrial membrane potential, and β-galactosidase activity with higher mRNA expression levels of apoptotic genes (Bax, cytochrome-c and caspase3) and a lower level of an anti-apoptotic gene (Bcl
2
) as compared to the control. Further, DEHP significantly (
P
> 0.05) decreased the level of steroid hormones (estradiol and progesterone) in a conditioned medium and this effect was reciprocated with a lower expression (
P
> 0.05) of steroidogenic responsive genes (Cyp11a1, Cyp19A1, Star, ERβ1) in treated granulosa cells. Furthermore, co-treatment with
N
-Acetyl-Cysteine (NAC) rescues the effects of DEHP on OS, ROS, β-galactosidase levels and gene expression activities. Altogether, these results suggest that DEHP induces oxidative stress
via
ROS generation and inhibits steroid synthesis
via
modulating steroidogenic responsive genes, which leads to the induction of apoptosis through the activation of Bax/Bcl-2-cytochrome-c and the caspase-3-mediated mitochondrial apoptotic pathway in rat granulosa cells.
Increased oxidative stress (OS) due to ubiquitous exposure to di-(2-ethylhexyl) phthalate (DEHP) can affect the quality of oocytes by inducing apoptosis and hampering granulosa cell mediated steroidogenesis.