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Two Virus Based Endocrine Disruptor Assays Effective Across Vertebrate Classes.
Citation:
Hartig, P., M. Cardon, K. Bobseine, E. Gray, AND V. Wilson. Two Virus Based Endocrine Disruptor Assays Effective Across Vertebrate Classes. Presented at American Society for Microbiology Annual Meeting, Denver, CO, May 18 - 21, 2013.
Impact/Purpose:
This study was used to validate two assay protocols
Description:
The presence of hormone mimics, or endocrine disrupting compounds (EDC’s), in the environment are increasing. Sources range from agricultural run–off, pharmaceuticals in waste water, to industrial operations. Current levels of contamination are sufficient to alter sexual development in exposed species and could pose a concern for potable water. Detecting these contaminants and setting safe exposures levels for both humans and wild life are compounded by the potential for differences in biological sensitivity across species. Herein, we focus on two virus-based assays used to assess chemicals/samples of androgenic and anti-androgenic activity across multiple species. Useful biologically based steroid hormone receptor assays have focused on receptor binding and transcriptional activation endpoints. Augmenting this approach we have developed a hormone binding assay based on baculovirus expressed, guanidine chloride (GnHCL) extracted, receptor and a transcriptional activation (TA) assay utilizing adenovirus transduction of primate cells. Exploiting published sequence data we have developed expression vectors capable of producing androgen receptor from at least one member of each vertebrate class without using animal tissue [African Clawed Frog (Xenopus laevis), Florida redbelly turtle (Pseudemys nelsoni), Fathead Minnow (Pimephales promelas), Chicken ( Gallus gallus) and the Chimpanzee (Pan troglodytes)]. Preliminary validation studies of the binding assay focused on the Chimp AR, with direct substitutions of other AR’s demonstrated. Ligand binding was easily assessed across species using extracted virus infected SF 21 cell lysates. Only the fish AR proved problematic due to GnHCL sensitivity. Dihydro- testosterone (DHT) was the test ligand for TA induction. TA specificity was demonstrated across all species. Preliminary data suggest DHT log EC50 values of -9.4 (chimp), -9.6 (fish), -10.6 (bird),-10.7 (frog) and -10.5 (turtle). The ability of the TA assay to detect anti-androgens was assessed for primate and fish receptors. Both accurately detected antagonists. Taken together these virus based assay provide a platform for assessing androgen receptor agonist and antagonist activity across all vertebrate classes. Abstract does not reflect US EPA policy.