Ligand-Mediated Receptor Assembly as an End Point for High-Throughput Chemical Toxicity Screening

Abstract

The high throughput screening of chemicals for interaction with intracellular targets is gaining prominence in the toxicity evaluation of environmental chemicals. We describe ligand-mediated receptor assembly as an early event in receptor signaling and its application to the screening of chemicals for interaction with targeted receptors. We utilized bioluminescence resonance energy transfer (BRET) to detect and quantify assembly of the methyl farnesoate receptor (MfR) in response to various high-production volume and other chemicals. The hormone methyl farnesoate binds to the MfR to regulate various aspects of reproduction and development in crustaceans. The MfR protein subunits Met and SRC, cloned from Daphnia pulex, were fused to the fluorophore, mAmetrine and the photon generator, Rluc2, respectively. Ligand-mediated receptor assembly was measured by photon transfer from the photon donor to the fluorophore resulting in fluorescence emission. Overall, the BRET assay had comparable or greater sensitivity as compared to a traditional reporter gene assay. Further, chemicals that screened positive in the BRET assay also stimulated phenotypic outcomes in daphnids that result from MfR signaling. We concluded the BRET assay is an accurate, sensitive, and cost/time efficient alternative to traditional screening assays.

A Crab Is Not a Fish: Unique Aspects of the Crustacean Endocrine System and Considerations for Endocrine Toxicology

“…The herbicide atrazine reportedly increased male offspring production in Daphnia pulicaria ( 299 ). However, atrazine did not interact with the MfR ( 37 ), suggesting that if atrazine did indeed activate this pathway, it may have been due atrazine increasing endogenous MF. Such an effect may have been the consequence of competition between atrazine and MF at a site of inactivation or elimination.…”

“…MET derives its name from the discovery that resistance of Drosophila to the insecticidal MF-analog, methoprene, was associated with a functional mutation in this gene ( 286 ). MF-activated MET recruits the bHLH-PAS protein, steroid receptor coactivator (SRC) ( 37 ). Together, this MET-SRC complex comprises the activated MF- receptor (MfR) in crustaceans.…”

“…The MfR is the best-demonstrated target of disruption of this regulatory pathway by environmental chemicals. Compounds that elicit insecticidal activity as juvenile hormone analogs also typically bind and activate the MfR ( 37 , 285 ). This activity is responsible for the high sensitivity of crustaceans to this class of insecticides ( 273 , 297 , 298 ).…”

“…However, the MfR appears to have high ligand recognition specificity. We are aware of no demonstrations that compounds, other than juvenile hormone analogs, are capable of activating the MfR at environmentally relevant exposure levels ( 37 ).…”

“…The generally high concentrations that are necessary to produce negative consequences for growth or reproduction suggest that the observed effects were merely a result of overt toxicity (e.g., (31,32). More specific assays are being developed that use elements of the crustacean endocrine system, such as reporter assays for the ecdysteroid and methyl farnesoate receptors, measurement of 20-hydroxyecdysone (20E) titers, or assays for chitobiase activity (33)(34)(35)(36)(37)(38). Nevertheless, establishing reliable testing protocols for endocrine disruption in crustaceans remains challenging and widely ignores the importance of neurohormonal regulation for the control of many physiological functions in crustaceans.…”