Triazine-mediated disruption of BLTK1 Leydig cell steroidogenesis
Triazines are broad spectrum herbicides implicated in the etiology of testicular dysgenesis eliciting perturbations of gonad development and reproductive function. In rodents triazines alter the onset of puberty, cause reproductive senescence, and disrupt reproductive tract development including dysregulation of androgen-dependent tissue development. Disruption of testosterone biosynthesis is thought to underlie the effects of triazines in males across vertebrate species. However, the current approaches used to identify developmental and reproductive toxicants involve in vivo studies evaluating apical endpoints that are generally descriptive and do not contribute to the elucidation of mechanism of action. As such, the mechanism by which triazines disrupt steroidogenesis remains unknown. The objectives of this study were to establish a model for the evaluation of triazines effects on steroidogenesis that is amenable to mechanism determination and to identify a possible mode of action for triazine-elicited disruption of steroidogenesis.BLTK1 Leydig cells were characterized and demonstrated to be a viable in vitro model for the evaluation of endocrine disruptor-mediated effects on steroidogenesis. BLTK1 cells express all necessary steroidogenic enzymes essential for hormone biosynthesis and maintain low basal levels of testosterone (T) production, inducible by recombinant human chorionic gonadotropin (rhCG) and forskolin (FSK). The time- and concentration-dependent effects of triazine herbicides, atrazine (ATR), propazine (PRO), simazine (SIM) and terbuthylazine (TBA), and their chlorometabolites, desethylatrazine (DEA), desisopropylatrazine (DIA) and diamino-s-chlorotriazine (DACT), were evaluated in BLTK1 Leydig cells. Triazines and their chlorometabolites induced concentration-dependent increases in basal progesterone (P) and T levels. Triazines also elicited the differential gene expression of several steroidogenic enzymes required for steroidogenesis. These results were consistent with the cumulative risk assessment "Common Mechanism Group" designation that PRO, SIM, DEA, DIA and DACT have ATR-like effects. Using ATR as the representative triazine, whole-genome microarrays identified differential gene expression at later time points (> 12 hr) with affected genes associated with steroidogenesis and cholesterol metabolism. Finally, the effects of ATR on rhCG-mediated induction of steroidogenesis revealed antagonism of P and T levels, despite potentiation of intracellular cAMP levels. The inhibition of cAMP-specific phosphodiesterases was identified as underlying increases in cAMP levels. However, the ATR-mediated super-induction of cAMP levels was not causative of T antagonism in the presence of rhCG. Disruption of phosphorylation cascades likely contribute to ATR-mediated effects on steroidogenesis, with observed effects on protein kinase A (PKA) target proteins. This study has established BLTK1 cells as a novel in vitro steroidogenic model for the evaluation of endocrine disrupting chemicals and the evaluation of triazines in BLTK1 cells has expanded our knowledge of triazine-mediated disruption of steroidogenesis.
Read
- In Collections
-
Electronic Theses & Dissertations
- Copyright Status
- In Copyright
- Material Type
-
Theses
- Authors
-
Forgacs, Agnes
- Thesis Advisors
-
Zacharewski, Timothy R.
- Committee Members
-
Henry, R. William
LaPres, John J.
Bursian, Steven J.
Vieille, Claire
- Date
- 2013
- Degree Level
-
Doctoral
- Language
-
English
- Pages
- xix, 160 pages
- ISBN
-
9781303298004
1303298007