Biosensing estrogenic endocrine disruptors in human blood and urine: A RAPID cell-free protein synthesis approach
Graphical abstract
Introduction
Unintentional as well as intentional discharge of harmful chemicals into the environment has been the conventional reality of industrialized society for hundreds of years. In recent decades, an increasing wealth of evidence has shown a certain class of chemicals known as endocrine disrupting chemicals (EDCs) to be of concern (Gore et al., 2015). Studies have detected significant levels of EDC activity in air (Holmes, 2016), soil (Aitkenhead et al., 2014), drinking water (Padhye et al., 2014), food (Minta et al., 2013), personal care products (De Coster and van Larebeke, 2012), pharmaceuticals (Scognamiglio et al., 2016), and synthetic hormones (Scognamiglio et al., 2016). These studies suggest that EDC exposure likely contributes to acute and chronic conditions including cancer (Soto and Sonnenschein, 2010), diabetes (Attina et al., 2016), obesity (De Coster and van Larebeke, 2012; Nalbone et al., 2013), metabolic syndrome (Philips et al., 2017), infertility (De Coster and van Larebeke, 2012), and permanent brain damage (De Coster and van Larebeke, 2012). A recent report estimated an EDC-exposure health burden of $340 billion USD in the United States and $209 billion USD in the EU (Attina et al., 2016).
One class of EDC's known as xenoestrogens (XEs) interferes specifically with the function of estrogen receptors. XEs originate from both natural (e.g. soy plants) and unnatural (e.g. BPA) sources. Research has linked exposure to XEs with obesity (Teixeira et al., 2015), birth defects (Titus-Ernstoff et al., 2010) including DNA methylation and placental alteration (Vilahur et al., 2014a), cancer (Morgan et al., 2016), reproductive impairment (N'Tumba-Byn et al., 2012), cognitive disabilities (Elsworth et al., 2015; Zwart et al., 2015), and developmental disorders (Vilahur et al., 2014b). Thus, public chemical safety would be enhanced with rapid, reliable, and cost-effective methods to screen chemicals, environmental samples, and human/animal samples for high levels of XE activity.
Characterizing XE interactions with estrogen receptors also benefits medical technology. Pharmaceuticals called selective estrogen receptor modulators are currently used to treat a variety of conditions including infertility, breast cancer, and postmenopausal complications, and are one of the World Health Organization's “essential medicines” (Moens et al., 2012; Johansen et al., 2013). Rapid screening technologies for ER modulators are valuable tools in drug discovery and characterization. Detection of estrogens and their derivatives in blood and urine samples is also an important diagnostic tool (Venners et al., 2006; Hormann et al., 2014).
Long-standing methods for detecting XEs utilize yeast and human cell lines (Soto et al., 1995; Routledge and Sumpter, 1996; Balaguer et al., 1999; Legler et al., 1999; Sonneveld et al., 2005; Leusch et al., 2010). While these are reliable and sensitive, their complicated laboratory procedures and long assay durations prohibit rapid screening and in-field detection (Alvarez et al., 2013; Scognamiglio et al., 2016; Conley et al., 2017). LC/MS and GC/MS are likewise popular techniques, but require trained technicians and significant equipment (~$190,000) (Ye et al., 2005; Tomaszewski et al., 2014; Covaci et al., 2015). Strategies employing biosensor proteins have been investigated in whole-cell (McLachlan et al., 2011) and purified-protein formats (De et al., 2005), but these methods require mammalian cell culturing and protein purification, both of which are cumbersome processes. There is a need for rapid and inexpensive methods for identifying XEs.
In a recent study, we introduced our Rapid Adaptable Portable In-vitro Detection biosensor platform (RAPID) for determining EDC activity (Salehi et al., 2017). This biosensor platform relies on exploiting the basic cellular mechanism of EDC activity in the following manner. An allosterically activated fusion protein containing the ligand-binding domain of a nuclear hormone receptor (NHR) and the reporter enzyme β-lactamase is synthesized in a cell-free protein synthesis (CFPS) reaction in the presence of a sample. If the sample contains NHR binding ligands, an increase in colorimetric signal is generated real-time. Unlike many emerging biosensor technologies, the RAPID biosensor is not analyte specific. Instead, the sensor detects binding interactions with a NHR.
Our previous work demonstrated the utility of this biosensor to detect and screen EDCs that interact with the Human thyroid receptor β (hTRβ). In this work, we demonstrate the modular nature of the RAPID biosensor by replacing the hTRβ domain with that of the human estrogen receptor β (hERβ) to detect XEs. The CFPS reaction allows direct utilization of the biosensor protein without cell culture or protein purification. We further engineer the CFPS biosensing reaction using RNAse inhibitors to achieve significantly enhanced protein synthesis, and thereafter XE detection, in human blood and urine. To our knowledge, this is the first report of biosensor proteins produced in CFPS reactions engineered to overcome adverse sample matrix effects.
Section snippets
Materials
All EDC ligands were purchased from Sigma-Aldrich (St. Louis, Missouri USA): diarylpropionitrile (DPN), bisphenol A (BPA), β-estradiol (E2), and TRIAC (3,3′,5-triiodothyroacetic acid). Blood samples were purchased from Innovative Research (Novi, Michigan, USA). The human donor urine was donated anonymously by the BYU health center. Nitrocefin was purchased from Cayman Chemical (Ann Arbor, Michigan USA), and murine RNAse inhibitor was purchased from New England Biolabs (Ipswich, Massachusetts).
Biosensor design and construction
RAPID biosensor design for the hERβ
The modular, flexible nature of the RAPID biosensor is demonstrated for the first time by adapting it to detect hERβ-specific endocrine disruptors. The RAPID biosensor, as illustrated in Fig. 1A, was constructed by replacing the human thyroid receptor β ligand-binding domain in our previously reported sensor (Salehi et al., 2017) with the hERβ ligand-binding domain.
Cell-free protein synthesis of the reporter fusion protein
The RAPID biosensor protein (Fig. 1A) was expressed using an E. coli-based CFPS system. The total protein production and solubility
Discussion
This work demonstrates that CFPS detection of hERβ ligands contributes several key advantages to estrogenic EDC detection technology. First, CFPS detection of estrogenic EDCs is rapid, requiring 2.5 h, while conventional cell-based biosensors require days or weeks of mammalian, yeast, or bacterial cell culturing before readout. The use of CFPS also removes cell culturing steps from the detection workflow and enables rapid execution. This characteristic makes the RAPID biosensor especially
Conclusion
Here we expanded the application of our RAPID biosensor to detect chemicals that interact with the human estrogen receptor β (XEs). Specifically, we demonstrated the modular, flexible nature of this biosensor can be exploited to expand the RAPID biosensor to additional nuclear hormone receptors and the detection of other types of endocrine disrupting chemicals. The RAPID biosensor has the same level of sensitivity as the analogous E. coli cell-based assays and many other reported cell-based
Conflict of interest statement
The authors report funding from NIH, NSF, and DARPA, and two patents pending regarding detection of endocrine disrupting chemicals using cell-free protein synthesis. The authors have no other conflicts of interest to declare.
Specifically:
Dr. Salehi reports grants from NSF, grants from DARPA, during the conduct of the study.
Dr. Yang reports grants from NSF, grants from DARPA, during the conduct of the study.
Conner Earl reports grants from NSF, grants from DARPA, during the conduct of the study.
Acknowledgments
The authors would like to thank the funding resources for their generous contribution including, NIH grant 1R21ES16630 to David Wood, NSF CAREER Award1254148 to Bradley Bundy, and DARPA Young Faculty AwardD13AP000037 to Bradley Bundy.
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These authors contributed equally to this work.