Identification of the herbicide, Oxyfluorfen, as an endocrine disrupting chemical using in vitro targeted screens and the Tier 1 Male Pubertal and Weanling Hershberger assays.
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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 (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 and weanling hershberger assays. 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 (PPS) was evaluated starting on PND 38 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 (3.3 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 evaluate the potential androgen antagonist effects, we exposed juvenile rats to oxyfluorfen in the male weanling hershberger assay. Animals were exposed to oxyfluorfen (62.5 and 125mg/kg) and testosterone propionate (1mg/kg) daily for 10 days. At 24 hours post last dose, serum was collected and androgen tissue weights were recorded. Oxyfluorfen significantly suppressed glans penis, seminal vesicle, and ventral prostate tissue growth as well as reduced serum T4 and T3 levels. 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, 0.200μM, 2μM, 10μM and 20μM and 125pM 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 and hershberger assays through two different mechanisms of action as shown by targeted in vitro screening assays. This abstract does not necessarily reflect U.S. EPA policy.