The current problem regards an ovarian site of action for 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin (TCDD) in modulation of female reproductive function. TCDD has been referred to as the most toxic substance known to man and is produced primarily as a by-product of waste incineration. Aside from acting as a teratogen, embryotoxin, or tumor progressor, TCDD is a known endocrine disrupter in laboratory species, wildlife, and from limited data, humans. We have demonstrated attenuated secretion of ovarian estrogen, and altered expression of ovarian mRNAs for estrogen- and dioxin- or aromatic hydrocarbon receptor (ER and AHR) in peri-pubertal rat pups after in-vivo administration of TCDD to pregnant dams. The evidence strongly supports the ovary as a major target of TCDD action. Because ovary (including granulose cells (GC) possesses AHR, we now propose uniquely to investigate the action of TCDD on human ovarian estrogen biosynthesis using an in-vitro culture paradigm. Our hypothesis is that TCDD exerts an anti-reproductive effect by binding AHR and diminishing ovarian steroidogenesis at one or more loci in steroid biosynthetic pathway, e.g., by modifying levels of P450 side-chain cleavage (SCC, CYP11A) or aromatase (CYP19) mRNA, protein and activity; and has no effect on estrogen-metabolizing enzymes (CYP1A1, 1B1).
Aim (1a). To determine effects of low-dose (environmentally relevant) TCDD on steroidogenic endpoints. We will administer TCDD to primary ovarian GC cultures from adult women; and characterize the effects on the levels of SCC mRNA using quantitative RT-PCR, activity by biochemical methods, and enzyme protein using Western immunoblotting; (1b) since our data in rat also show modulation of P450 aromatase enzyme, we will evaluate the endpoints in (1a) above for this enzyme; (2) unexpected but conceivable alternative to reduced estrogen synthesis is ovarian metabolism of estrogen via cytochromes P450. We will evaluate levels of mRNAs for ovarian CYP1A1 and 1B1, which preferentially catalyze estrogens (estradiol and estrone) to their 2-OH- and 4-OH metabolites, respectively; and separate any products chromatographically, in order to assess whether both reduced synthesis and catabolism might occur simultaneously in the ovary. Collectively, these mechanistic molecular studies will point to the ovary as a major target of TCDD action, and will aid us in determining the basis for the reproductive dysfunction observed with dioxin in animals and humans. This AREA will serve as a """"""""seed grant"""""""" to produce preliminary data and assist us in deciding the course of action to pursue in a R01 application.
