**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].

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 [6–8], rely on expensive analytical equipment [8, 9], or do not meet the U.S. Environmental Protection Agency's (EPA)

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Realizing the deficiencies of these screening programs, and receiving considerable pressure from the public to reduce the use of animals for EDC testing, the U.S. EPA, with stakeholder input from the NIH National Institute of Environmental Health Sciences (NIEHS), has established the Endocrine Disruptor Screening Program for the twenty-first century (EDSP21) [10]. The goal of EDSP21 is to replace the current battery of Tier 1 tests with less expensive and faster high-throughput assays that can reduce the number of compounds that unnecessarily move forward to Tier 2 testing. This focus on improving the characterization of chemicals during Tier 1 screening is paramount to controlling costs, as mischaracterizations (i.e., false positives) during the Tier 1 stage magnify the costs of downstream Tier 2 screening, with a chemical's progression through multiple phases of Tier 2 screening only to be classified as negative for EDC activity representing a very poor return on investment. With the current chemical backlog approaching 87,000 chemicals [11], and considering the conventional scientifically acceptable false positive error rate of 5%, under current Tier 1 testing formats a minimum of 4350 chemicals will likely mistakenly proceed toward Tier 2 screening at a cost

The use of autobioluminescent EDC cellular bioreporters represents an attractive means to overcome the limitations of existing Tier 1 screening platforms and address the needs of the EDSP21 program. Autobioluminescence, with is defined as the ability to self-initiate the production of a luminescent signal using only endogenously supplied substrates to perform the enzymatic reactions necessary for signal generation [12], can reduce the number of required assay steps, eliminate the need for superfluous reagent costs, maintain human bioavailability relevance through the use of human cellular hosts, and increase throughput by minimizing hands-on performance time and employing automated processing and detection systems [13]. These benefits are made possible by the autonomous functionality of the synthetic luciferase gene cassette (*lux*) that controls the autobioluminescent phenotype. To enable autonomous EDC detection, *lux* cassette expression is regulated by a yeast upstream activating sequence (UAS), which is itself activated by a hybrid Gal4 transcriptional activator. Expression of this activator is, in turn, governed by the binding of an EDC to

full testing requirements [3].

of approximately \$8 billion.

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].

**Figure 1.** Tens of thousands of chemicals are suspected of having the potential to interfere with the endocrine system, resulting in adverse health effects in people and wildlife.

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 [6–8], rely on expensive analytical equipment [8, 9], or do not meet the U.S. Environmental Protection Agency's (EPA) full testing requirements [3].

**1. Introduction**

128 Endocrine Disruptors

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

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

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].

**Figure 1.** Tens of thousands of chemicals are suspected of having the potential to interfere with the endocrine system,

plasticizers, number in the tens of thousands (**Figure 1**) [2].

normal endocrine function in humans and wildlife.

resulting in adverse health effects in people and wildlife.

Realizing the deficiencies of these screening programs, and receiving considerable pressure from the public to reduce the use of animals for EDC testing, the U.S. EPA, with stakeholder input from the NIH National Institute of Environmental Health Sciences (NIEHS), has established the Endocrine Disruptor Screening Program for the twenty-first century (EDSP21) [10]. The goal of EDSP21 is to replace the current battery of Tier 1 tests with less expensive and faster high-throughput assays that can reduce the number of compounds that unnecessarily move forward to Tier 2 testing. This focus on improving the characterization of chemicals during Tier 1 screening is paramount to controlling costs, as mischaracterizations (i.e., false positives) during the Tier 1 stage magnify the costs of downstream Tier 2 screening, with a chemical's progression through multiple phases of Tier 2 screening only to be classified as negative for EDC activity representing a very poor return on investment. With the current chemical backlog approaching 87,000 chemicals [11], and considering the conventional scientifically acceptable false positive error rate of 5%, under current Tier 1 testing formats a minimum of 4350 chemicals will likely mistakenly proceed toward Tier 2 screening at a cost of approximately \$8 billion.

The use of autobioluminescent EDC cellular bioreporters represents an attractive means to overcome the limitations of existing Tier 1 screening platforms and address the needs of the EDSP21 program. Autobioluminescence, with is defined as the ability to self-initiate the production of a luminescent signal using only endogenously supplied substrates to perform the enzymatic reactions necessary for signal generation [12], can reduce the number of required assay steps, eliminate the need for superfluous reagent costs, maintain human bioavailability relevance through the use of human cellular hosts, and increase throughput by minimizing hands-on performance time and employing automated processing and detection systems [13]. These benefits are made possible by the autonomous functionality of the synthetic luciferase gene cassette (*lux*) that controls the autobioluminescent phenotype. To enable autonomous EDC detection, *lux* cassette expression is regulated by a yeast upstream activating sequence (UAS), which is itself activated by a hybrid Gal4 transcriptional activator. Expression of this activator is, in turn, governed by the binding of an EDC to

**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 proceeds similarly.

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**.

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

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

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

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

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.

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

EC50—half maximal effective concentration. EC50—half maximal effective concentration.

EDC detection with high human relevance [6–9, 14].

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 EDSP21 program.
