The effect of dichlorodiphenyldichloroethylene (DDE) on steroidogenesis in granulosa cells
Date
2000
Authors
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ORCID
Type
Degree Level
Masters
Abstract
The insecticide dichlorodiphenyltrichloroethane (DDT) and its major metabolite
p,p'- dichlorodiphenyldichloroethylene (DDE) are endocrine disrupters. The DDT
metabolite p,p'-DDE has been found contaminating human tissues and follicular fluid
around the world, due to its persistence and continued use. The focus of this research
was to investigate the effects of DDE on progesterone synthesis in a stable porcine
granulosa cell line, JC-410, and in primary cultures of porcine granulosa cells. Further
objectives of this research were to validate the JC-410 cell line as an in vitro model for
the study of endocrine disrupters on ovarian steroidogenesis, and to elucidate the
mechanism of action of DDE.
Low concentrations of DDE, 0.1-100 ng/ml, did not affect basal progesterone
synthesis in the JC-410 cells. However, 10 ng/ml DDE increased 8-Br-cAMP- and
cholera toxin (CT)-stimulated progesterone synthesis 0.4-0.7-fold over the levels
observed with 8-Br-cAMP or CT alone. This effect was confirmed in primary cultures
of porcine granulosa cells. Neither basal nor CT-stimulated cAMP levels were changed
by 0.1-100 ng/ml DDE. In the JC-410 cells, 1 and 10 ng/ml DDE increased CT stimulated
cytochrome P450-cholesterol side chain cleavage (P450scc) mRNA levels
0.3- and 0.4-fold over the values observed with CT alone.
High concentrations of DDE, 3000 and 10000 ng/ml, reduced basal progesterone
synthesis in the JC-410 cells 0.51- and 0.75-fold, respectively. As well, DDE, 300-10000 ng/ml, blocked the CT-induced stimulation of progesterone synthesis. High
concentrations of DDE, 3000 and 10000 ng/ml, decreased basal cAMP generation 0.33-
and 0.64-fold, respectively. The addition of DDE 300, 3000, and 10000 ng/ml decreased CT-stimulated cAMP levels by 0.16, 0.32, and 0.48-fold respectively, as
compared to CT alone. Basal expression of the P450scc gene was decreased 0.61- and
0.82-fold by 3000 and 10000 ng/ml DDE, respectively. Addition of DDE, 3000 and
10000 ng/ml, blocked the CT-induced stimulation of P450scc gene expression. Cellular
protein levels were increased 0.35- and 0.42-fold, respectively, by 3000 and 10000
ng/ml DDE.
It was concluded that DDE, at environmentally relevant concentrations, modulates
granulosa cell steroidogenesis in a dose dependent manner, through two different
mechanisms of action. At low concentrations, DDE potentiates the conversion of
cholesterol to pregnenolone induced by PKA activators, thus stimulating progesterone
production. At high concentrations, DDE inhibits basal and stimulated cAMP
generation and P450scc gene expression, thus decreasing progesterone production.
Based on these observations, it is possible that DDE could disrupt gonadotropin control
of ovarian steroidogenesis, thus representing a threat to human reproductive health and
fertility.
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Degree
Master of Science (M.Sc.)
Department
Toxicology
Program
Toxicology