**4. Autobioluminescent detection assay formats**

#### **4.1. Advantages**

validate that there are sufficient ER concentrations and to confirm that the receptor is functioning with appropriate affinity. Only after this series of preliminary steps are the actual assays run. During the assay, radioactively labeled 17β-estradiol, uterine cytosol, and test chemical are combined and must undergo a 16–20 h incubation at 4°C in the dark. Following incubation, hydroxyapatite is added, and multiple washings are performed before a final elution with ethanol and measurement of radioisotope activity in a liquid scintillation counter [7]. Similarly, the complementary androgen receptor (AR) binding assay follows the same intricate assay steps as the ER binding assay, but begins with the collection of rat ventral prostate tissues using subjects that are castrated ~24 h prior to assay initiation. Similar to the ER binding assay, this inclusion of approximately 19 animal subjects per test chemical results

Like the ER and AR binding assays, the aromatase assay also uses radioactively labeled chemicals as detection targets. In addition, assay performance also requires the use of controlled substances, and therefore necessitates specialized waste disposal. Although these attributes do not directly hinder assay performance, they add cost and increase the logistical hurdles underlying assay execution. However, the tradeoff for the use of these chemicals is an increased throughput. Under standard conditions, the aromatase assay can be completed with only 6–8 person hours per run. This makes the aromatase assay a more attractive format for companies concerned with personnel costs. Another advantage of the aromatase assay is that it uses human recombinant microsomes as the detection vehicle, which provides additional human bioavailability relevance compared with the use of animal tissues in the ER and

Unlike the above-mentioned assays, the steroidogenesis assay uses a human adrenocortical carcinoma cell line as its detection vehicle, which provides direct information on the human-relevant effects of compound exposure. However, while this represents a significant advantage, it also comes with the drawback that the cells must remain exposed to the test

in increased moral, economical, and logistical concerns [6].

**Table 3.** The five traditional *in vitro* tier 1 EDC detection assays used in EDSP21.

AR binding assays [8].

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Autobioluminescent assays systems address the backlogging problems endemic to EDSP21 because they utilize human cellular hosts as their detection vehicles, their signal generation is fully performed by these host cells without the need for external stimulation, their resulting reporter signal does not require cellular destruction or interfere with cellular metabolism, they are capable of self-regulating bioluminescent production throughout EDC exposure, and they maintain the same output format (luminescent production) as the commonly used ERTA [13]. Because these same output and detection vehicle formats are maintained, autobioluminescent assay systems share the advantages of providing direct human bioavailability information and not requiring specialized equipment or skilled personnel to perform. However, unlike the ERTA, the bioreporter cells used in autobioluminescent assays do not require lysis and therefore remain viable for an unlimited number of repeated or fully continuous measurements. This allows cytotoxicity measurements to be taken on control wells within each plate at any time point desired and eliminates the need for duplicate plate preparation. Since all data are obtained in real-time, the assay intervals employed in autobioluminescent assays can be shortened or lengthened on-the-fly based on the results being obtained, which provides an increased level of flexibility when working with previously uncharacterized compounds (**Figure 3**). In addition, the detection equipment used to perform the ERTA can be used to perform autobioluminescent assays, so no change in equipment infrastructure is required [17].

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

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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

**Figure 4.** Using repeated measurements of T47-D breast cancer cell line samples, the autobioluminescent assay format allowed dose/response relationships between autobioluminescence and EDC treatment levels to be determined for each

day that showed a significant increase compared to negative control cells.

controls (**Figure 5A**).

This continuous imaging ability of autobioluminescence provides higher levels of data acquisition than the alternative assay formats and is more amenable to high-throughput use. This results in a significant cost savings of approximately 87% per assay relative to the ERTA, which has the lowest performance costs of the alternative assay types. For example, under moderate throughput conditions a 96-well microtiter test plate can be used to accommodate triplicate replicates of four test chemicals and their associated controls. Using this testing format, it would require 21,750 96-well plates to characterize the existing backlog of 87,000 chemicals that are pending under EDSP21 [11]. Based on existing market costs for technician time and chemical reagents [18, 19], it would cost approximately \$1.5 billion (USD) to process all of these compounds. However, the reduced performance costs of the autobioluminescent assay format, which result primarily from a reduction in technician hands-on time and removal of the need to purchase an activating chemical substrate, reduces these costs to approximately \$191,000 (USD), representing a savings of approximately \$1.3 billion (USD).

#### **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

**Figure 3.** (A) The continuous signal generation of autobioluminescent assays allows for uninterrupted, real-time, highthroughput monitoring of cell activity across consecutive time points. This increases flexibility relative to (B) the ERTA, which only generates single time point snapshots of cellular activity.

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**).

(**Figure 3**). In addition, the detection equipment used to perform the ERTA can be used to perform autobioluminescent assays, so no change in equipment infrastructure is required [17]. This continuous imaging ability of autobioluminescence provides higher levels of data acquisition than the alternative assay formats and is more amenable to high-throughput use. This results in a significant cost savings of approximately 87% per assay relative to the ERTA, which has the lowest performance costs of the alternative assay types. For example, under moderate throughput conditions a 96-well microtiter test plate can be used to accommodate triplicate replicates of four test chemicals and their associated controls. Using this testing format, it would require 21,750 96-well plates to characterize the existing backlog of 87,000 chemicals that are pending under EDSP21 [11]. Based on existing market costs for technician time and chemical reagents [18, 19], it would cost approximately \$1.5 billion (USD) to process all of these compounds. However, the reduced performance costs of the autobioluminescent assay format, which result primarily from a reduction in technician hands-on time and removal of the need to purchase an activating chemical substrate, reduces these costs to approximately

\$191,000 (USD), representing a savings of approximately \$1.3 billion (USD).

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

**Figure 3.** (A) The continuous signal generation of autobioluminescent assays allows for uninterrupted, real-time, highthroughput monitoring of cell activity across consecutive time points. This increases flexibility relative to (B) the ERTA,

**4.2. Performance and EDC detection abilities**

134 Endocrine Disruptors

which only generates single time point snapshots of cellular activity.

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

**Figure 4.** Using repeated measurements of T47-D breast cancer cell line samples, the autobioluminescent assay format allowed dose/response relationships between autobioluminescence and EDC treatment levels to be determined for each day that showed a significant increase compared to negative control cells.

**Figure 5.** (A) An autobioluminescent MCF-7 cell line was capable of fully autonomous 17β-estradiol detection but displayed a low signal-to-noise ratio. (B) Re-created HEK293 cell line expressing human estrogen receptor alpha and the autobioluminescent reporter construct allowed for fully autonomous 17β-estradiol detection with an improved signalto-noise ratio.

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**).

**5. Future directions and recommendations**

relative to alternative tier 1 screening methods.

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

**Table 6.** Summary of the observed advantages and disadvantages of the autobioluminescent EDC detection format

**Table 4.** When expressed in HEK293 cells, the estrogen compound-responsive autobioluminescent reporter system

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**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.

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.

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**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.


**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**

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**). 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.

to-noise ratio.

136 Endocrine Disruptors

**Figure 5.** (A) An autobioluminescent MCF-7 cell line was capable of fully autonomous 17β-estradiol detection but displayed a low signal-to-noise ratio. (B) Re-created HEK293 cell line expressing human estrogen receptor alpha and the autobioluminescent reporter construct allowed for fully autonomous 17β-estradiol detection with an improved signal-

> 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.

EC50 Half maximal effective concentration

century

EPA U.S. Environmental Protection Agency

ERTA Estrogen receptor transactivation assay

EDSP21 Endocrine Disruptor Screening Program for the twenty-first

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ICCVAM Interagency Coordinating Committee on the Validation of

NIEHS NIH National Institute of Environmental Health Sciences

, Jody Toperzer2

1 Center for Environmental Biotechnology, The University of Tennessee, Knoxville, USA

[1] Schug T, Janesick A, Blumberg B, Heindel J. Endocrine disrupting chemicals and disease susceptibility. Journal of Steroid Biochemistry and Molecular Biology. 2011;

, Marvin Steven Furches2

, Steven Ripp1

,

LC/APPI-MS/MS Liquid chromatography positive atmospheric pressure photoion ization tandem mass spectroscopy

PC10 Concentration inducing a response at 10% of the maximal positive

PC50 Concentration inducing a response at 50% of the maximal positive

Alternative Methods

control response

control response

EDC Endocrine disruptor chemical

ER Estrogen receptor

ERE Estrogen response element

*lux* Synthetic luciferase gene cassette

PBS Phosphate buffered saline

, Andrew Kirkpatrick2

and Dan Close2

2 490 BioTech, Knoxville, Tennessee, USA

**Author details**

Tingting Xu1

Gary Sayler2

**References**

**127**(3-5):204-215

UAS Upstream activating sequence

\* \*Address all correspondence to: dan.close@490biotech.com
