Open access peer-reviewed chapter

# Rapid, High-Throughput Detection of Endocrine Disrupting Chemicals Using Autobioluminescent Cellular Bioreporters

By Tingting Xu, Andrew Kirkpatrick, Jody Toperzer, Marvin Steven Furches, Steven Ripp, Gary Sayler and Dan Close

Submitted: February 26th 2018Reviewed: May 7th 2018Published: November 5th 2018

DOI: 10.5772/intechopen.78378

## Abstract

Overexposure to endocrine disruptor chemicals (EDCs) can result in serious health problems, yet they are commonly found in everyday items such as pesticides, personal care products, nutritional supplements, and plastics. The U.S. Environmental Protection Agency, along with other such agencies from around the world, have therefore mandated that new approaches be designed to screen these products for the presence of EDCs. However, despite the presence of several types of extant EDC detection assays, there still exists a backlog approaching 87,000 chemicals currently awaiting screening. Autobioluminescent detection systems, which utilize cellular bioreporters capable of autonomously modulating bioluminescent signals without the need for external stimulation or investigator interaction, provide an attractive means for addressing this backlog because of their reduced performance costs and increased throughput relative to alternative assay systems. This chapter reviews the variety of existing EDC detection assays and evaluates the performance of a representative autobioluminescent estrogen-responsive EDC bioreporter to provide an overview of how autobioluminescence can be used to improve EDC detection using in vitro assay systems.

### Keywords

• bioreporter
• autobioluminescence
• high-throughput analysis
• endocrine disruptor
• estrogen
• luciferase

## 1. Introduction

The human endocrine system is an interconnected, finely tuned network of glands that produce hormones responsible for health and well-being from the time of conception until death. Chemicals classified as endocrine disruptors (EDCs) interfere with the production, release, transport, and/or action of these hormones and cause imbalances that are suggested to result in significant negative health impacts such as infertility, premature puberty, obesity, diabetes, heart disease, and breast, prostate, testicular, thyroid, endometrial, and ovarian cancers [1]. These chemicals, which are present in a variety of sources including pesticides, cosmetics, and plasticizers, number in the tens of thousands (Figure 1) [2].

The potential adverse effects of EDCs on human, wildlife, and ecosystem health have received significant worldwide attention from the scientific community, regulatory agencies, and the general public. Unfortunately, the uncertainties inherent to understanding the true health consequences of EDC exposure have fostered significant controversy, and the lay person is besieged with an extensive collection of ‘facts’ when attempting to grasp the fundamental content of the EDC problem. One only needs to Google bisphenol-A (BPA) to appreciate the informational complexity surrounding a chemical suspected of being an endocrine disruptor. Capitalizing on the difficulties posed by this situation, a multitude of companies have formed to evaluate how the compounds that make up everyday items such as pesticides, personal care products, nutritional supplements, and plastics can imbalance the delicate regulation of normal endocrine function in humans and wildlife.

There are currently over 500 contract testing service companies in the U.S. alone that are dedicated to performing assays for the chemical, pesticide, and personal care products industries, and this industry is expected to continue growing year-over-year at an annual rate of 13.5% [3]. To improve throughput and decrease costs, these companies have adapted a two-tiered screening format, with Tier 1 consisting of in vitro assays aimed at identifying those chemicals that have the potential to interact with the endocrine system, and Tier 2 re-screening those compounds that test positive using in vivo assays to define their endocrine-related effects and obtain dosage-relevant information. Unfortunately, despite their societal importance, these tests remain biologically, logistically, and economically challenging. Tier 1 testing of chemicals for potential EDC activity is estimated to cost from $100,000 to$250,000 per chemical, with Tier 2 testing requiring upwards of 1,200 experimental animals and costing $1.2–$2.5 million per chemical [4, 5]. The majority of these costs will be borne by the chemical manufacturing industry, which then trickles down as increased prices at the consumer level. Furthermore, many of the common Tier 1 assay formats employed by these companies use non-human cell lines that can obscure bioavailability data [6, 7], require the use of radioactive materials that necessitate dedicated use areas and specially trained personnel [6, 7, 8], rely on expensive analytical equipment [8, 9], or do not meet the U.S. Environmental Protection Agency’s (EPA) full testing requirements [3].

### 4.2. Performance and EDC detection abilities

To evaluate the utility of autobioluminescence’s repeated interrogation approach, autobioluminescent T47-D cells were seeded in triplicate into multi-well plates and incubated under standard growth conditions for 24 h. After this time, the medium was removed, cells were washed once with phosphate buffered saline (PBS), refreshed with EDC-free medium, and supplemented with 17β-estradiol at concentrations of 0 pM (control), 0.1 pM, 1 pM, 10 pM, 100 pM, 1 nM, 10 nM, or 100 nM. Autobioluminescent measurements were then obtained every 24 h for 6 days using an IVIS Lumina imaging system with a 10 min integration time. Increased autobioluminescent signals relative to untreated control cells were observed by day 3 for all treatments ≥1 pM, although this trend was only maintained throughout the full 6 day assay period at treatment levels ≥10 pM. A dose–response relationship was observed between 17β-estradiol treatment levels and autobioluminescence, with an EC50 value of 10 pM (Figure 4). Similar results were obtained using the alternative MCF-7 breast cancer cell line, which could detect 17β-estradiol at concentrations of both 1 and 10 nM through the significant (p < 0.05) induction of an autonomously-regulated autobioluminescent signal compared to both background light detection and the signal generated cells treated only with vehicle controls (Figure 5A).

Notably, the autobioluminescent production from both of these breast cancer cell lines displayed a relatively low signal-to-noise ratio, likely due to their natural expression of estrogen receptors and EDC transporters. To overcome this limitation, the system was re-created in the naturally ER-negative HEK293 human kidney cell line and co-transfected with human estrogen receptor alpha. This allowed for expression of the system without interference from native EDC uptake and processing pathways and significantly reduced the level of background autobioluminescent production in the absence of EDC stimulation, as well as increasing the signal-to-noise ratio during positive detection events (Figure 5B). Using this system design, EDC-responsive autobioluminescent HEK293 bioreporters were able to detect an array of representative EDCs at levels relevant to the requirements of EDSP21 (Table 4).

### Table 4.

When expressed in HEK293 cells, the estrogen compound-responsive autobioluminescent reporter system detected an array of representative EDCs within the EPA detection guidelines.

This bioreporter similarly proved to be effective for the detection of other commonly encountered EDCs, such as synthetic hormones, synthetic industrial compounds, phytoestrogens, and fungicides (Table 5). These detection capabilities are especially promising given that the autobioluminescent system can be scaled to allow for robotic integration. This would allow cell plating, dosing, and reading to be fully automated. Since the addition of exogenous substrate or sample manipulation post-treatment is not required, this system reduces assay complexity and facilitates rapid detection using automated systems. Given its advantages relative to the existing assay formats (Table 6), autobioluminescence represents an attractive alternative assay for potential high-throughput Tier 1 screening of the EPA’s current chemical inventory list.

### Table 5.

The autobioluminescent HEK293-based estrogenic compound-responsive bioreporter was found to be an efficient and simplistic means for the detection of a wide variety of compounds with known estrogenic effects.

### Table 6.

Summary of the observed advantages and disadvantages of the autobioluminescent EDC detection format relative to alternative tier 1 screening methods.

## 5. Future directions and recommendations

While autobioluminescent assays have the potential to significantly improve the throughput and cost effectiveness of Tier 1 EDC detection, they are currently in their infancy. Of the tested methods, only the HEK293-based autobioluminescent assay format was capable of producing data with similar performance metrics to the incumbent screening procedures. It is clear that the utility of the autobioluminescent assay format will need to expand to additional cell types and to the detection of androgenic compounds in order to fully address the bioavailability and health effects of EDCs. Similarly, while this work screened the performance of the HEK293-based estrogen-responsive bioreporter against a variety of EDCs and associated controls, it will be necessary to validate the performance of this assay format at the levels of scale required for commercial use. Therefore, the development of additional bioreporter cell types and their validation at scale using automated assay preparation, performance, and detection equipment is recommended as a next step in the maturation of this assay format. If autobioluminescent assays can perform reliably under these conditions while maintaining a similar level of performance to that observed from the HEK293-based estrogen-responsive bioreporter, they will prove a valuable tool for Tier 1 EDC detection.

## 6. Conclusions

Tier 1 in vitro assays are the front line in EDC detection. However, the limitations of traditional assay formats, which use non-human cell lines that can obscure bioavailability data [6, 7], require the use of radioactive materials that necessitate dedicated use areas and specially trained personnel [6, 7, 8], or rely on expensive analytical equipment [8, 9], are currently incapable of handling the sheer number of compounds that must be screened. Autobioluminescent assays, such as the HEK293-based estrogen-responsive bioreporter assay presented here, are uniquely positioned to overcome the limitations of existing assay formats by autonomously generating bioluminescence in response to target chemical or chemical class bioavailability. The use of these reporter systems allows bioluminescent responses to be linked to EDC detection for reagent-free, fully automated screening at a fraction of the cost of existing assays, providing a promising route toward addressing the existing EDC compound screening backlog.

## Acknowledgments

The authors acknowledge research funding provided by the U.S. National Institutes of Health under Award Numbers NIEHS-1R43ES022567-01, NIEHS-2R44ES022567-02, and NIEHS-1R5ES023979-01.

## Conflict of interest

S.R., G.S., and D.C. are board members in the for-profit entity 490 BioTech.

## Abbreviations

 AR Androgen receptor ARE Androgen response element BPA bisphenol-A EC50 Half maximal effective concentration EDC Endocrine disruptor chemical EDSP21 Endocrine Disruptor Screening Program for the twenty-first century EPA U.S. Environmental Protection Agency ER Estrogen receptor ERTA Estrogen receptor transactivation assay ERE Estrogen response element ICCVAM Interagency Coordinating Committee on the Validation of Alternative Methods LC/APPI-MS/MS Liquid chromatography positive atmospheric pressure photoionization tandem mass spectroscopy lux Synthetic luciferase gene cassette NIEHS NIH National Institute of Environmental Health Sciences PC10 Concentration inducing a response at 10% of the maximal positive control response PC50 Concentration inducing a response at 50% of the maximal positive control response PBS Phosphate buffered saline UAS Upstream activating sequence

chapter PDF
Citations in RIS format
Citations in bibtex format

## More

© 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution 3.0 License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

## How to cite and reference

### Cite this chapter Copy to clipboard

Tingting Xu, Andrew Kirkpatrick, Jody Toperzer, Marvin Steven Furches, Steven Ripp, Gary Sayler and Dan Close (November 5th 2018). Rapid, High-Throughput Detection of Endocrine Disrupting Chemicals Using Autobioluminescent Cellular Bioreporters, Endocrine Disruptors, Ahmed R. G., IntechOpen, DOI: 10.5772/intechopen.78378. Available from:

### Related Content

#### Endocrine Disruptors

Edited by Ahmed R.G.

Next chapter

#### Occurrence of Endocrine Disruptor Chemicals in the Urban Water Cycle of Colombia

By Diego Fernando Bedoya-Ríos and Jaime Andrés Lara-Borrero

#### Growth Disorders and Acromegaly

Edited by Ahmed R.G.

First chapter

#### Introductory Chapter: Growth Disorders

By Ahmed R.G.

We are IntechOpen, the world's leading publisher of Open Access books. Built by scientists, for scientists. Our readership spans scientists, professors, researchers, librarians, and students, as well as business professionals. We share our knowledge and peer-reveiwed research papers with libraries, scientific and engineering societies, and also work with corporate R&D departments and government entities.

View all Books