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EFFECTS OF ATRAZINE ON STEROID PRODUCTION IN RAT GRANULOSA CELLS
Citation:
Tinfo, N., M. G. Hotchkiss, R. L. COOPER, AND S. C. LAWS. EFFECTS OF ATRAZINE ON STEROID PRODUCTION IN RAT GRANULOSA CELLS. Presented at Society for the Study of Reproduction, Kona, HI, May 28 - 31, 2008.
Impact/Purpose:
Presentation at SSR
Description:
Atrazine is one of the most widely used herbicides in the United States. Introduced in the 1950s, atrazine is a broad spectrum herbicide with current total annual use of approximately 76 million pounds of active ingredient. Frogs exhibit gonadal malformations and/or variations in normal hormone production following exposure to atrazine. Based on in vitro studies using H295R cells, it has been speculated that these effects are the result of the influence of atrazine on aromatase (enzyme responsible for the conversion of androgens into estrogens) expression and/or activity; however, this hypothesis remains controversial. In order to determine if/how atrazine is affecting aromatase activity and reproductive function, the specific molecular mechanisms which are targeted by atrazine need to be determined. The orphan nuclear receptor steroidogenic factor 1 (SF-1) regulates the transcription of a large number of genes involved in steroidogenesis and reproduction. SF-1 binds its response element and regulates transcription constitutively; however, ligand binding may enhance SF-1 activity. It has been shown that atrazine increases SF-1 expression, and binds to SF-1. Since SF-1 is located in the rat aromatase promoter, and has been localized to rat granulosa and theca cells, atrazine may induce binding and activation of the aromatase promoter, leading to an increase in aromatase activity. The current studies were conducted to determine if aromatase expression and activity could be measured in rat granulosa cells in culture and to determine the effects of atrazine on these measures. Twenty-seven day old Wistar rats were injected with 20 IU of PMSG to stimulate follicular development. After 48 hours the rats were killed and ovaries collected. Granulosa cells were isolated and placed in culture with vehicle alone (0.3 % ethanol), atrazine (1, 10 or 30 µM), testosterone (0.017 µM), or 4-hydroxyandrostenedione (10 µM). Following 24 hours in culture some of the media and granulosa cells were collected for radio-immunoassays (RIAs) and real time PCR, respectively, and other cells remained in culture to measure aromatase activity using the tritiated water assay. Atrazine at all three concentrations did not affect estradiol production; however, atrazine at 10 and 30 µM significantly increased progesterone concentrations as compared to controls (p ≤ 0.05). Aromatase (CYP19) and SF-1 expression of mRNA were not significantly affected by any of the atrazine treatments. In summary, our results show that it is possible to detect aromatase activity from granulosa cells in culture: however, it has yet to be determined if atrazine affects aromatase activity in granulosa cell cultures. These results suggest that atrazine does not affect follicular estrogen production, but that it may affect luteinization or luteal hormone production.