Biosensing estrogenic endocrine disruptors in human blood and urine: A RAPID cell-free protein synthesis approach

https://doi.org/10.1016/j.taap.2018.02.016Get rights and content

Highlights

  • Cell-free protein synthesis (CFPS) detection of estrogenic endocrine disruptors

  • CFPS in blood and urine improved with addition of RNAse inhibitors.

  • Rapid CFPS detection of the E2 in human blood and urine

Abstract

Many diseases and disorders are linked to exposure to endocrine disrupting chemicals (EDCs) that mimic the function of natural estrogen hormones. Here we present a Rapid Adaptable Portable In-vitro Detection biosensor platform (RAPID) for detecting chemicals that interact with the human estrogen receptor β (hERβ). This biosensor consists of an allosteric fusion protein, which is expressed using cell-free protein synthesis technology and is directly assayed by a colorimetric response. The resultant biosensor successfully detected known EDCs of hERβ (BPA, E2, and DPN) at similar or better detection range than an analogous cell-based biosensor, but in a fraction of time. We also engineered cell-free protein synthesis reactions with RNAse inhibitors to increase production yields in the presence of human blood and urine. The RAPID biosensor successfully detects EDCs in these human samples in the presence of RNAse inhibitors. Engineered cell-free protein synthesis facilitates the use of protein biosensors in complex sample matrices without cumbersome protein purification.

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.

References (54)

  • V. Scognamiglio et al.

    Analytical tools monitoring endocrine disrupting chemicals

    Trac-Trends Anal. Chem.

    (2016)
  • G. Skretas et al.

    A bacterial biosensor of endocrine modulators

    J. Mol. Biol.

    (2005)
  • M.T. Smith et al.

    Alternative fermentation conditions for improved Escherichia coli-based cell-free protein synthesis for proteins requiring supplemental components for proper synthesis

    Process Biochem.

    (2014)
  • N. Vilahur et al.

    Prenatal exposure to mixtures of xenoestrogens and repetitive element DNA methylation changes in human placenta

    Environ. Int.

    (2014)
  • N. Vilahur et al.

    In utero exposure to mixtures of xenoestrogens and child neuropsychological development

    Environ. Res.

    (2014)
  • J.H. Zhang et al.

    A simple statistical parameter for use in evaluation and validation of high throughput screening assays

    J. Biomol. Screen.

    (1999)
  • A. Blank et al.

    Ribonucleases of human serum, urine, cerebrospinal fluid, and leukocytes. Activity staining following electrophoresis in sodium dodecyl sulfate-polyacrylamide gels

    Biochemistry

    (1981)
  • L. Cevenini et al.

    A novel bioluminescent NanoLuc yeast-estrogen screen biosensor (nanoYES) with a compact wireless camera for effect-based detection of endocrine-disrupting chemicals

    Anal. Bioanal. Chem.

    (2017)
  • S. De Coster et al.

    Endocrine-disrupting chemicals: associated disorders and mechanisms of action

    J. Environ. Public Health

    (2012)
  • J.D. Elsworth et al.

    Low circulating levels of bisphenol-a induce cognitive deficits and loss of asymmetric spine synapses in dorsolateral prefrontal cortex and hippocampus of adult male monkeys

    J. Comp. Neurol.

    (2015)
  • M.D. Gawrys et al.

    Use of engineered Escherichia coli cells to detect estrogenicity in everyday consumer products

    J. Chem. Technol. Biotechnol.

    (2009)
  • I. Gierach et al.

    Bacterial biosensors for evaluating potential impacts of estrogenic endocrine disrupting compounds in multiple species

    Environ. Toxicol.

    (2013)
  • A.C. Gore et al.

    Executive summary to EDC-2: the Endocrine Society's second scientific statement on endocrine-disrupting chemicals

    Endocr. Rev.

    (2015)
  • D. Holmes

    Endocrine disruptors: air pollution linked to insulin resistance

    Nat. Rev. Endocrinol.

    (2016)
  • A.M. Hormann et al.

    Holding thermal receipt paper and eating food after using hand sanitizer results in high serum bioactive and urine Total levels of bisphenol a (BPA)

    PLoS One

    (2014)
  • J.P. Hunt et al.

    The growing impact of lyophilized cell-free protein expression systems

    Bioengineered

    (2017)
  • M. Iwama et al.

    Purification and properties of human urine ribonucleases

    J. Biochem.

    (1981)
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    These authors contributed equally to this work.

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