**2. Requisite endocrine disrupting chemical detection parameters**

The U.S. EPA [14] and the Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM) [15] have established performance requirements for all EDC detection assays. These performance requirements ensure that the assays can function efficiently enough to identify the presence of EDCs at levels believed to be impactful to human and environmental health. However, rather than mandating that an assay demonstrates predetermined responses


PC50—concentration which induces a response at 50% of the maximal positive control response.

PC10—concentration which induces a response at 10% of the maximal positive control response.

EC50—half maximal effective concentration.

EC50—half maximal effective concentration.

an upstream estrogen (ERE; pictured) or androgen (ARE) response element (**Figure 2**). The use of this EDC-responsive promoter system within a human cell can therefore signal EDC bioavailability while simultaneously providing information regarding the timing, magnitude, and duration of the resulting effect. Using the detection of estrogenic compounds as an example, this chapter will provide an overview of how these autobioluminescent cellular bioreporters function in this role relative to alternative, traditional Tier 1 EDC sensor platforms and the advantages and disadvantages they provide for addressing the needs of the

**Figure 2.** Functional schematic of an estrogenic compound-responsive autobioluminescent induction system. Step 1: *Gal4ff* expression is induced upon estrogenic compound exposure. Step 2: The *lux* cassette is then activated through stimulation of the UAS/TATA promoter by the *Gal4FF* transcriptional activator. Androgenic compound induction

**2. Requisite endocrine disrupting chemical detection parameters**

The U.S. EPA [14] and the Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM) [15] have established performance requirements for all EDC detection assays. These performance requirements ensure that the assays can function efficiently enough to identify the presence of EDCs at levels believed to be impactful to human and environmental health. However, rather than mandating that an assay demonstrates predetermined responses

EDSP21 program.

proceeds similarly.

130 Endocrine Disruptors

**Table 1.** EPA guidelines for the successful detection of endocrine disruptor chemicals in human cells.


**Table 2.** ICCVAM average EC50 value guidelines required for the successful detection of androgenic compounds.

across all known EDC compounds, these organizations require that the assay respond appropriately to treatment with serial dilutions of representative strong, weak, and very weak agonists, and that they do not respond to an appropriate negative control. To be considered successful, estrogenic detection assays must meet the U.S. EPA metrics presented in **Table 1** and androgenic detection assays must meet the ICCVAM metrics presented in **Table 2**.

#### **3. Non-autobioluminescent detection assay formats**

There are five *in vitro* assay formats, other than autobioluminescence, that are used for EDSP21 Tier 1 screening [16] (**Table 3**). All of these assays are well-established, having been initially developed as early as the 1960's. Although their performance is reproducible and reliable, each is subject to a number of detriments that limit their utility for low-cost, high-throughput EDC detection with high human relevance [6–9, 14].

For instance, in the estrogen receptor (ER) binding assay, cytosol must be isolated from the uteri of rats that have undergone ovariectomy prior to collection of the uterine tissue. These animal subjects are ovariectomized 7–10 days before harvesting the uterine tissue, with each test chemical requiring the use of approximately 19 subjects. Once the uteri have been harvested, they are homogenized and centrifuged to isolate ER-containing cytosol. Before conducting the assay, saturation radioligand binding assays using various concentrations of radioactively labeled 17β-estradiol added to each batch of cytosol are performed to first


chemical for 48 h, making this one of the longer duration assay formats. Further complicating the throughput of the assay is the detection method, which uses liquid chromatography positive atmospheric pressure photoionization tandem mass spectroscopy (LC/APPI-MS/MS) to measure the hormone concentrations in the medium as the assay endpoint. While this provides exquisite levels of sensitivity, the equipment required to perform these measurements is relatively expensive and requires highly-skilled technical personnel for operation. This limits the performance of this assay format to only those labs large enough to justify the associated

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The estrogen receptor transactivation assay (ERTA), also uses a human cell line as its detection vehicle. In this case, the assay leverages a human cervical cancer cell line containing a firefly luciferase reporter gene that emits a bioluminescent signal when chemicals bind to and activate the estrogen receptor. The bioluminescent output of this format makes it an attractive option because it does not require specialized equipment or skilled personnel to perform. The cells for this assay are simply plated in microtiter plates, the test chemical is added, and the plates are incubated for 20–24 h. Following incubation, the luciferase assay reagent is then added to each well to lyse the cells, and bioluminescence is measured. While this assay format is among the most simplistic to perform, the multi-day performance period and the need for requisite sample destruction concurrent with the addition of an exogenous activation chemical impart concerns relating to throughput, performance costs, and the potential interaction of the activating chemical with the compound under study. Nonetheless, the ETRA remains a popular choice for EDC

detection due to its many advantages relative to the alternative assay formats [14].

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

**4. Autobioluminescent detection assay formats**

operational costs [9].

**4.1. Advantages**

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

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

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 AR binding assays [8].

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 chemical for 48 h, making this one of the longer duration assay formats. Further complicating the throughput of the assay is the detection method, which uses liquid chromatography positive atmospheric pressure photoionization tandem mass spectroscopy (LC/APPI-MS/MS) to measure the hormone concentrations in the medium as the assay endpoint. While this provides exquisite levels of sensitivity, the equipment required to perform these measurements is relatively expensive and requires highly-skilled technical personnel for operation. This limits the performance of this assay format to only those labs large enough to justify the associated operational costs [9].

The estrogen receptor transactivation assay (ERTA), also uses a human cell line as its detection vehicle. In this case, the assay leverages a human cervical cancer cell line containing a firefly luciferase reporter gene that emits a bioluminescent signal when chemicals bind to and activate the estrogen receptor. The bioluminescent output of this format makes it an attractive option because it does not require specialized equipment or skilled personnel to perform. The cells for this assay are simply plated in microtiter plates, the test chemical is added, and the plates are incubated for 20–24 h. Following incubation, the luciferase assay reagent is then added to each well to lyse the cells, and bioluminescence is measured. While this assay format is among the most simplistic to perform, the multi-day performance period and the need for requisite sample destruction concurrent with the addition of an exogenous activation chemical impart concerns relating to throughput, performance costs, and the potential interaction of the activating chemical with the compound under study. Nonetheless, the ETRA remains a popular choice for EDC detection due to its many advantages relative to the alternative assay formats [14].
