In vitro confirmation of estrogenic and antiandrogenic pesticide bioactivity using schild regression analysis

A broad range of anthropogenic chemicals have been reported to display estrogenic (ER) or antiandrogenic bioactivity using high throughput screening (HTS) in vitro assays. Some regulatory agencies have developed tiered in vitro – in vivo endocrine screening batteries in which positive in vitro results automatically “trigger” studies with laboratory animals. Since in vitro assays can produce a number of false positive and false negative results, automatically triggering in vivo testing could result in the unnecessary use of animals and other resources. The in vitro false positive rate may be particularly high with reported AR antagonists, because many nonspecific mechanisms can disrupt competitive AR dose-response assays such that chemicals falsely appear to be competitive AR ligands. In the current investigation, we illustrated the utility of in vitro Schild regression to interrogate the in vitro ER and/or anti-AR bioactivity of pesticides which were positive in HTS ER or AR models. Schild regression discriminates chemicals that act as true competitive receptor ligands from those that disrupt signaling via noncompetitive mechanisms. The chemicals studied included seven pesticides listed by EPA as high priority for in vivo ER or anti-AR testing and two pesticides listed as low priority, as well as 17β-estradiol (E2) and hydroxyflutamide (OHF) as ER and anti-AR reference ligands. Two out of four pesticides tested for ER agonist activity were cytotoxic, and four out of seven pesticides tested for AR antagonist activity, plus OHF, were true competitive AR antagonists (two true positives and two false negatives). Herein, we propose a tiered strategy that includes a more in-depth analysis of in vitro bioactivity using Schild regression to determine if HTS or other in vitro bioactivity data results from true competitive receptor antagonism or some nonspecific mechanism. This strategy could reduce unnecessary in vivo testing for chemicals that are not AR antagonists in vitro.