Identification of the herbicide, Oxyfluorfen, as an endocrine disrupting chemical using in vitro targeted screens and the Tier 1 Male Pubertal assay.
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Identification of the herbicide, Oxyfluorfen, as an endocrine disrupting chemical using in vitro targeted screens and the Tier 1 Male Pubertal assay.
Murr, AS1, A. Buckalew1, Bailey, J.3, Ford, J.2 and Stoker, TE1.
1Neurological and Endocrine Toxicology Branch, PHITD, CPHEA, 2CCTE, ORD, US EPA, RTP, NC. 3Oak Ridge Institute for Science and Education, US DOE, Oak Ridge, TN.
Oxyfluorfen is a pre- and post-emergent diphenyl-ether herbicide used to control weeds in tree, fruit, and nut crops, as well as residential areas. Recently, we identified oxyfluorfen as a potent inhibitor of iodide uptake, the first step in the synthesis of thyroid hormones, using two in vitro (human and rat) sodium iodide symporter (NIS) radioactive iodide uptake assays. Here, we further evaluate the effects of oxyfluorfen on thyroid hormone synthesis in vivo using the EDSP Tier 1 male rat pubertal assay. Oxyfluorfen was administered by daily oral doses of 0, 15, 31.25, 62.5, 125, 250, and 500mg/kg in 1% methyl cellulose vehicle for 31 days (PND 23 to PND 54) to juvenile Sprague-Dawley rats. Daily body weights were collected during dosing and preputial separation was evaluated starting on PND 36 until completion. Two hours following the last dose on PND 54, trunk blood was collected for serum hormone and chemical analysis. Oxyfluorfen exposure significantly suppressed serum T4 and T3 at all doses (15 to 500 mg/kg) with maximum suppression of 80 and 60 %, respectively. No significant effects were observed on serum pituitary hormones (thyroid stimulating or luteinizing hormone). Interestingly, oxyfluorfen also delayed male reproductive androgen dependent tissue growth and pubertal progression. There was a significant delay in preputial separation (1.7 to 3.6-day delay at 31.25 to 500 mg/kg) and a suppression of ventral prostate and seminal vesicle growth at the two highest doses, but with no change in testes weight or serum testosterone. No effects on growth or final body weight were observed. There were no changes on kidney or adrenal weights, but relative liver weights were significantly increased from at doses of 62.5 and higher. To further evaluate the antagonistic effects of oxyfluorfen, we conducted an in vitro rat androgen receptor (AR) transcriptional activation reporter assay (Indigo Biosciences kit) using oxyfluorfen concentrations of 0, 200nM, 2μM, 10μM and 20μM and 125 pM 11-ketotestosterone (11-kt) in 0.2% DMSO for a 24-hour incubation period. Oxyfluorfen showed significant concentration-dependent inhibition of luminescence activity at 2 to 20 µM in the presence of the agonist 11-kt, demonstrating AR antagonism. This study demonstrated that oxyfluorfen suppressed both the production of thyroid hormones and pubertal progression in the pubertal assay through two different mechanisms of action as shown by targeted in vitro screening assays. This abstract does not necessarily reflect U.S. EPA policy.