Toxicology in the Fast Lane: Application of High-Throughput Bioassays to Detect Modulation of Key Enzymes and Receptors

Background Legislation at state, federal, and international levels is requiring rapid evaluation of the toxicity of numerous chemicals. Whole-animal toxicologic studies cannot yield the necessary throughput in a cost-effective fashion, leading to a critical need for a faster and more cost-effective toxicologic evaluation of xenobiotics. Objectives We tested whether mechanistically based screening assays can rapidly provide information on the potential for compounds to affect key enzymes and receptor targets, thus identifying those compounds requiring further in-depth analysis. Methods A library of 176 synthetic chemicals was prepared and examined in a high-throughput screening (HTS) manner using nine enzyme-based and five receptor-based bioassays. Results All the assays have high Z′ values, indicating good discrimination among compounds in a reliable fashion, and thus are suitable for HTS assays. On average, three positive hits were obtained per assay. Although we identified compounds that were previously shown to inhibit a particular enzyme class or receptor, we surprisingly discovered that triclosan, a microbiocide present in personal care products, inhibits carboxylesterases and that dichlone, a fungicide, strongly inhibits the ryanodine receptors. Conclusions Considering the need to rapidly screen tens of thousands of anthropogenic compounds, our study shows the feasibility of using combined HTS assays as a novel approach toward obtaining toxicologic data on numerous biological end points. The HTS assay approach is very useful to quickly identify potentially hazardous compounds and to prioritize them for further in-depth studies.

Although pharmaceuticals and pesticides are evaluated for toxicity at great cost, numer ous anthropogenic compounds produced in sizable amounts and present in our everyday environment have not been tested for any tox icologic activity. The recent California Green Chemistry Report (California Department of Toxic Substances Control 2008) illustrates that far more chemicals are in common use than the ones tested for toxicity, and in most cases, there are few or no toxicity data for a large number of these chemicals. Novel inter national legislation, such as the Registration, Evaluation, Authorization and Restriction of Chemicals (REACH) program implemented in 2007 by the European Union (European Chemicals Agency 2007), requires that all chemicals used in the European Union at more than 1 metric ton/year/company be evaluated for their toxicity over the next decade. Ultimately, the European Union may develop an authorization system to control substances of very high concern and pro gressively replace them with suitable alter natives where economically and technically viable, unless there is an overall benefit for society of using the substance. The U.S. Environmental Protection Agency has sev eral voluntary programs, including the High Production Volume Challenge Program (U.S. Environmental Protection Agency 1998), that allow compiling of chemical toxicity and haz ard information for selected chemicals. It is very likely that additional national and inter national legislation will be enacted that will require generation of toxicity data for most of the chemicals produced in sizable quantity.
For almost 200 years, laboratory animal testing has been the major tool of toxicologists (Gad 2006). However, such tests have the dis advantages of being both timeconsuming and very costly because they require use of large number of animals, and they are not always predictive of human risk. For the implemen tation of REACH, Scialli (2008) estimated that tens of million of animals will be used at a cost of several hundred thousand dollars per compound, making it very challenging to use experimental animals to complete analy sis of the toxicologic effects of many chemi cals in a reasonable time frame. Accordingly, there is a need for accurate toxicologic eval uation of xenobiotics to be faster and more costeffective. Progress in molecular biology, biotechnology, and other fields have paved the way for toxicity testing to be quicker, less expensive, and more directly relevant to human exposures (Gibb 2008). Although it is certain that in vitro assays cannot yet replace animal testing (Tingle and Helsby 2006), they may provide essential information that can prioritize and dramatically reduce the use of animal testing assays (Silliman and Wang 2006). However, when considering the pros pect of screening tens of thousands of chemi cals against hundreds of in vitro assays, several important questions need to be answered. Can enzyme or cellbased bioassays yield useful toxicologic information? Furthermore, can these assays be conducted in a highthrough put and reliable fashion, allowing the rapid screening of thousands of compounds for bio logical and toxicologic activities?
As part of the University of California-Davis Superfund Basic Research Program, whose aim is to identify biomarkers of expo sure and effects of toxic substances, we have developed a library of techniques, including numerous enzyme and cellbased screening assays (Ahn et al. 2008;Garrison et al. 1996;Han et al. 2004;Huang et al. 2007;Jones et al. 2005;Nagy et al. 2002;Rogers and Denison 2000;Shan and Hammock 2001). Although such assays are routinely used to find novel small chemical inhibitors in the pharmaceuti cal industry, we tested whether such mecha nistically based screening assays can be used to rapidly provide information on the potential for compounds to produce specific biological toxic effects that would identify those requiring further indepth study. More specifically, we tested whether these assays could be adapted for highthroughput screening (HTS). We Background: Legislation at state, federal, and international levels is requiring rapid evaluation of the toxicity of numerous chemicals. Whole-animal toxicologic studies cannot yield the necessary throughput in a cost-effective fashion, leading to a critical need for a faster and more cost-effective toxicologic evaluation of xenobiotics. oBjectives: We tested whether mechanistically based screening assays can rapidly provide information on the potential for compounds to affect key enzymes and receptor targets, thus identifying those compounds requiring further in-depth analysis. Methods: A library of 176 synthetic chemicals was prepared and examined in a high-throughput screening (HTS) manner using nine enzyme-based and five receptor-based bioassays. results: All the assays have high Z´ values, indicating good discrimination among compounds in a reliable fashion, and thus are suitable for HTS assays. On average, three positive hits were obtained per assay. Although we identified compounds that were previously shown to inhibit a particular enzyme class or receptor, we surprisingly discovered that triclosan, a microbiocide present in personal care products, inhibits carboxylesterases and that dichlone, a fungicide, strongly inhibits the ryanodine receptors. conclusions: Considering the need to rapidly screen tens of thousands of anthropogenic compounds, our study shows the feasibility of using combined HTS assays as a novel approach toward obtaining toxicologic data on numerous biological end points. The HTS assay approach is very useful to quickly identify potentially hazardous compounds and to prioritize them for further in-depth studies. key words: bioassays, biomarkers, enzyme inhibition, high-throughput assays, triclocarban, triclosan.  a small (176 compounds) and structur ally very diverse library from among commonly encountered environmental chemicals. We report the results of screening this library with nine enzymebased and five receptorbased bio assays. These assays were selected because the proteins involved were shown to interact with xenobiotics, and because the in vitro effects of these xenobiotics could be related to the in vivo activity of these proteins and health effects.

Materials and Methods
A more detailed account of the materials and methods used is given in the Supplemental Materials, (doi:10.1289/ehp.0900834.S1 via http://dx.doi.org/).
Chemicals. Most chemicals used in the library were from commercial sources. Chemicals were at least 95% pure and used without further purification.
Environmental chemicals library. The library was prepared in 2mL deepwell poly propylene 96well assay plates. Every com pound was dissolved at 10 mM in dimethyl sulfoxide (DMSO). Only compounds totally soluble at 10 mM in DMSO were included in the library. In each plate, the wells in the first column contained only DMSO to serve as controls. In the remainder of the plate, we dispensed one compound per well, with 88 compounds total per plate. We created two plates for a total of 176 compounds. A detailed description of the chemical contents in each plate is presented in the Supplemental Materials, Tables 1 and 2 (doi:10.1289/ ehp.0900834.S1). The sealed plates were stored at -20°C until use. Upon use, the plates were diluted to the appropriate concentration using a robotic pipetting station.
Enzyme assays. Although the condi tions for each enzyme assay were different (for details, see Table 1), the enzymatic assays were all run in a similar format. Enzymes were used at a concentration that results in linear generation of product with increasing time and protein concentration, as well as yielding a signal that was 3-20 times greater than the background. BSA (0.1 mg/mL final concentration) was added to all buffers just before use to reduce nonspecific inhibition (McGovern et al. 2002). For glutathione Stransferase (GST) activities, the buffer was supplemented with 5 mM glutathione. For all the enzyme assays, we tested the compounds at final concentrations of 0.1 and 1 µM.
Kinetic assay conditions. The dissociation constant of triclosan for CES1 was determined following the method described by Dixon (1972) for competitive tight binding inhibi tors, using cyano(6methoxy2 naphthyl) methyl acetate (CMNA) as the substrate (Shan and Hammock 2001). Inhibitor concentra tions between 0 and 1,000 nM were incubated in triplicate for 5 min in sodium phosphate buffer (pH 7.4) at 30°C with 200 µL of the enzyme solution. Substrate at a final concen tration of 5-100 µM was then added. Velocity of the reaction was measured as described above. For each substrate concentration, plots of velocity as a function of inhibitor concen tration allow the determination of an appar ent inhibition constant (K Iapp ). The plot of K Iapp as a function of the substrate concentra tion allows the determination of K I when the substrate concentration is zero. Results were expressed as the mean ± SD of three separate K I measurements.
Cell-based bioassays. Table 2 presents an overview of the different cellbased bioassays used. For all test compounds, agonist activ ity in the aryl hydrocarbon receptor (AhR), androgen receptor (AR), and estrogen recep tor (ER) assays was determined in the AhR, AR, and ER CALUX (chemically activated luciferase expression) bioassays, respectively. All three CALUX bioassays make use of differ ent cell lines (H1L6.1c2, T47DAR-positive, and BG1Luc4E2/ERα-positive, respectively) that contain a stably transfected luciferase gene under the transcriptional control of DNA response elements for the activated AhR, AR, and ER, respectively (Garrison et al. 1996;Han et al. 2004;Rogers and Denison 2000). Activation of the receptor signaling pathway was determined by quantifying the luciferase activity in the absence or presence of a known agonist [2,3,7,8tetrachloro dibenzopdioxin (TCDD), 17βestradiol (E 2 ), or dihydrotestosterone (DHT)]. Results were expressed relative to luciferase activity maximally induced by a reference compound (1 nM TCDD for AhR, 10 nM DHT for AR, 1 nM E 2 for ER). For these assays, the primary screening of the library was done at 10 µM. Membranes enriched in ryanodine receptors (RyRs) were obtained either from  adult rabbit skeletal muscle, a pure type 1 ryanodine receptor (RyR1) source (Saito et al. 1984), or from cardiac ventri cular tissue, a pure type 2 ryanodine receptor (RyR2) source (Pessah et al. 1990). Activation or inhibition of the receptors was measured by quantifying the ability of the tested compound at 5 µM to enhance or inhibit the basal binding of [ 3 H]Ry (2 nM) in the presence of 20 µM CaCl 2 . After a 3hr incubation at 37°C, the reactions were quenched by filtration through GF/Bgrade glass fiber filters and washed twice with ice cold harvest buffer containing 20 µM CaCl 2 .
[ 3 H]Ry binding was quantified by measuring the radioactivity collected on the filter. Selection of positive hits and counterscreening. For the enzyme assays, a compound was selected as a positive hit if it resulted in > 50% inhibition at the lower concentration (100 nM) and if it resulted in more than 60% inhibition at the higher concentration (1 µM). For the cellbased assays, we selected com pounds that significantly (ttest and Ftest, p < 0.01) induced the receptor activation of gene expression. For counterscreening, fresh solutions of all positive compounds were pre pared in DMSO. For the enzyme assays, the concentration of each compound that inhib ited 50% of the enzyme activity (IC 50 ) was determined by measuring enzyme activities in the absence and presence of increasing con centrations of inhibitor (ranging from 0.5 to 10,000 nM). IC 50 values were calculated by nonlinear regression of at least five data points using SigmaPlot, version 9.01 (Systat Software Inc., Chicago, IL). Results are provided as the mean ± SD of at least three separate measure ments. Similarly, halfmaximal effective con centration (EC 50 ) values for agonists of the AhR and ER bioassays were determined, and the results are presented as the mean of tripli cate analysis. For the assay of [ 3 H]Ry binding to RyR1 or RyR2, the influence of 5 µM of each compound was screened for its ability to either enhance or inhibit specific radioligand binding more than twice the baseline (defined as the level of [ 3 H] Ryspecific binding in the presence of DMSO alone). Therefore, a positive hit on RyR1 or RyR2 was defined as ≥ 200% of control binding for activators, or ≤ 50% of control for inhibitors.

Results and Discussion
Assays characteristics and positive hits selection. Using results from the blank and full activity controls, we evaluated the suitability of each assay for use as HTS assays. We there fore calculated the signaltobackground ratio (S/B), the signaltonoise ratio (S/N), and the Z´ factor as defined by Zhang et al. (1999). As shown in Table 3, we found that S/B ratios varied from 2.5 to > 150, with the lowest value for the absorbancebased assay (GSTs) and the highest for the radioactivebased assays (RyRs). Similarly, the S/N ratios varied greatly, with a lower value for the absorbance assay and the higher values for the radioactive based assays. In general, the enzymebased assays yielded higher Z´ factors than did the cellbased bioassays. For the enzyme assays, Z´ values were > 0.7, indicating very good and reliable assays that are easily suitable for HTS assays. Although the cellbased assays yielded lower Z´ factors, the values were still > 0.5, suggesting that the discrimination is adequate and that these assays could be used in HTS assays. Nevertheless, for the RyR assays, a larger separation band and higher Z´ factor could be obtained by reducing the SD of the signal, which was around 20%.
The aims of the primary screening were to identify all possible positive hits and to ensure there were no false negatives. Thus, for the primary screening of the library, we tested the xenobiotics at relative high concentrations (0.1 and 1 µM for the enzymes, and 5 and 10 µM for the receptors), which should be far higher than blood concentrations resulting from exposure. Thus, it is unlikely that com pounds found negative in the primary screen ing will be false negative and affect the tested proteins in vivo. Generally, testing higher con centrations result in solubility problems for an increasing proportion of compounds. Based on our definition of positive hits (described above), for the 14 assays we obtained a total of 69 positive results (Table 3), which rep resent on average five positive hits per assay, or 3% of the library. For FAAH, GST, and AR bioassays, we obtained no hits from the screening. There were twice as many posi tive hits from chemicals in plate II (42) than from those in plate I (27) [see Supplemental Material, Figure 1 (doi:10.1289/ehp.0900834. S1)]. The latter plate contained numerous triazine herbicides that did not result in any significant inhibition in any assay. Although three compounds [carbophenothion, triclosan, and triphenyl phosphate (TPP)] gave positive results with three enzymes or more, all the target enzymes were esterases.
Even if the assays are of high quality, as defined by their S/B, S/N, and Z´ factors (described above), false positives are bound to happen as they are dependent on the com pounds tested and not on the assays. False pos itives are mostly due to nonspecific binding, alteration of the reporting signal (quenching of the fluorescence signal, cytotoxicity to the cells, etc.), and chemical modifications dur ing storage of the chemicals. The purpose of the counterscreening is to eliminate false posi tives. To reduce nonspecific inhibition, BSA (0.1 mg/mL final concentration) was added to all buffers just before use (McGovern et al. 2002). To eliminate alteration of the report ing signal, we tested the ability of each positive hit to quench the fluorescent or luminescent signal as well as its possible cytotoxic effect. Unfortunately, it is not possible to run such controls in the primary screen format. Finally, to reduce false positives resulting from some chemical modification upon storage, we pre pared a fresh solution of each positive hit just before counterscreening. Out of the 69 positive hits initially found in the library screening, individual counterscreening analysis confirmed that 39 of them are effectively positive hits (see definition above), indicating an approxi mately 40% falsepositive rate for the primary screening. This relatively high number of false positives reflects the high concentrations used for the primary screening. A lower screening concentration will have a lower number of false positives but will signi ficantly increase the chance of false negatives, which is not desirable. Overall, using this twostep screening method, we found that 98% of the compounds tested have no effects on the tested assays.

Individual enzymes and receptors results.
For all the positive hits selected from the library screening, we determined their individual  (Table 4), except for the RyR assays, which are the sub ject of a forthcoming study. As expected, we found that sEH was strongly inhibited by two ureacontaining compounds, which are a well established class of sEH inhibitors (Morisseau et al. 1999): siduron and triclo carban [trichloro carbanilide (TCC)]. Although siduron uses are limited, TCC is present in numerous personal care products (Ahn et al. 2008), suggesting a large exposure risk. Animal models have shown that inhibition of the sEH affects human health by altering homeo stasis, blood pressure, inflammation, and pain (Morisseau and Hammock 2008). Inhibition of the CESs by organo phosphate xenobiotics (Table 4), such as carbo phenothion, parathion, phosdrin, and TPP, was expected, because such compounds are common mech anistic suicide inhibitors of serine hydrolases after activation to the oxon form (Casida and Quistad 2005). Because the CESs are only slowly reactivated, there is thus a cumulative risk. Although many organophosphate insecti cides have been or are being phased out around the world, TPP continues to be used both as a plasticizer and a fire retardant in electronic com ponents. Burning or leaching of TPP from elec tronic waste could result in its presence in water (Owens et al. 2007). Given the role of CES in the metabolism of ester and amidecontaining xenobiotics (Satoh and Hosokawa 2006), CES inhibition could lead to increased toxicity of xenobiotics. In general, CES inhibitors contain a carbonyl that reacts with the activesite serine to form a tetrahedral intermediate (Harada et al. 2009). Thus, the inhibition of CES1 and CES2 by triclosan, present in numerous personal care products (Ahn et al. 2008), was unexpected. To understand the mechanism of action of tri closan, we determined its kinetic constant [see Supplemental Material, Figure 2 (doi:10.1289/ ehp.0900834.S1)]. We found that triclosan inhibits CES1 by a competitive mechanism and a K I of 105 ± 5 nM. Although not the most potent of known CES1 inhibitors, triclosan represents a lead compound for a new class of esterase inhibitors.
PON2 was first identified as an enzyme that protects humans from environmental poison ing by organophosphate derivatives (James 2006); thus, one could expect apparent inhibition of this enzyme by organophosphates as we observed (Table 4). For carbophenothion and tributyl phosphotri thioite, this is likely due to traces of oxon impurities. Interestingly, we found that, in addition to CES1 and CES2, TPP can also significantly reduce PON2 activity. Inhibition of PON2 could lead to increased atherosclerosis and cardiovascular risk (James 2006). Taken together, exposure to TPP could affect human health through various modes of action.
For the two cytochrome P450 (CYP450) acti vities tested, significant inhibition was observed only for CYP450 2C9 (Table 4). 2Methylheptyl4,6dinitrophenyl crotonate, the active ingredient in the fungicide dinocap, was the only very potent inhibitor of this CYP450 found. Interestingly CYP450 2C9 is involved in the production of antiinflamma tory and antihypertensive epoxyeicosatrienoic acids from arachidonic acid; thus, inhibition of this CYP450 could lead to increased cardio vascular risk (Morisseau and Hammock 2008).
Screening results for the three nuclear receptor signaling pathways (AhR, ER, and AR) identified seven compounds with sig nificant agonist activity: two for AhR, five for ER, and none for AR. Interestingly, even given the promiscuity of AhR ligand binding (Denison and HeathPagliuso 1998;Denison and Nagy 2003), only two fungicide chemi cals, 2(4chlorophenyl)benzothiazole (CPB) and dichlone, induced AhRdependent gene expression, and they were relatively weak inducers. CPB and dichlone EC 50 values for induction (Table 4) were approximately 5 × 10 5 fold less potent than the proto typical AhR agonist TCDD. Although dichlone is a newly identified AhR agonist, CPB was pre viously reported to induce AhRdependent expression of cytochrome CYP450 1A1 in human and mouse cell lines (Kärenlampi et al. 1989). As expected, we found that ER sig nal transcription was activated by o,p´DDT (dichlorodiphenyltrichloroethane) and its metabolites o,p´DDE (dichlorodiphenyl dichloroethylene) and o,p´DDD (dichloro diphenyldichloroethane) (Chen et al. 1997;Rogers and Denison 2000), and our screen ing identified o,p´DDE and o,p´DDD as activators also (o,p´DDT was not pres ent in the screened library). In our system, the EC 50 for induction by o,p´DDE and o,p´DDD was approximately 10 5 fold less potent than that of E 2 (Rogers and Denison 2000). Similarly, bisphenol A (BPA) and lin dane have also been previously identified as ER agonists (BonefeldJørgensen et al. 2007;Maranghi et al. 2007;Steinmetz et al. 1996;Vandenberg et al. 2009), although lindane has been suggested to activate ERdependent gene expression through a nonclassical mechanism (Steinmetz et al. 1996). BPA was the most potent ER agonist identified, only 3,000fold less potent than E 2 , whereas lindane was the weakest. Taken together, the relatively low potency of these agonists coupled with exist ing controversies regarding exposure and health risks associated with BPA and other endocrinedisrupting chemicals (Vandenberg et al. 2009) suggests that the adverse effects of these chemicals remain to be determined.
Our primary screen revealed that numer ous compounds affected the RyRs, such as tri closan, which we previously showed to increase [ 3 H]Ry binding to RyR1 (Ahn et al. 2008). For counterscreening, we concentrated on the 12 chemicals that produced the most signifi cant RyR effect (Figure 1). Overall, the profiles for both receptors are similar, with the profile of RyR2 being more attenuated than that for RyR1. For the latter protein, we found eight compounds (at 5 µM) that significantly affected the binding of [ 3 H]Ry: five of them inhibited the binding, and three increased it. For RyR2, we found four compounds that significantly inhibited this receptor. For both receptors, the largest effect was observed for chloranil (IC 50 < 1.0 µM) and dichlone (IC 50 < 1.0 µM), which both contain in their structure a 2,3di chloro1,4quinone. These results are consistent with our previously published work showing that naphthoquinones and benzoquinones are capable of selectively modifying RyR1 chan nels in a time and concentrationdependent manner (Feng et al. 1999). Interestingly, we found that [ 3 H]Ry binding to RyR1 was increased almost 3fold by chlorpyriphos and o,p´DDE. Counterscreening results suggested that baythroid, αcypermethrin, delta methrin, and Ncyclohexyl2benzothiazyl sulfonamide have no significant effect on either RyR at 5 µM. Obtaining a compound that interacts specifically with only one of the RyRs or has opposing effects on both proteins will be scien tifically very important. The deltamethrin scaf fold could be a lead toward such compounds, because deltamethrin seemed to have oppos ing effects on both RyRs. RyR1 and RyR2 are major components of skeletal and cardiac muscle excitation contraction coupling, and several heritable mutations in these proteins have been associated with myogenic disor ders (Bellinger et al. 2008). In addition, RyR1 and RyR2 are the major isoforms expressed in neurons and are responsible for producing temporally and spatially defined Ca 2+ signals important for neuronal growth and plasticity (Berridge 2006). Deregulation of RyR func tion and expression contributes to alterations in activitydependent dendritic growth and plasticity (Kenet et al. 2007;Roegge et al. 2006;Yang et al. 2009) and the balance of excitatory and inhibitory neurotransmission in the hippocampus CA1 region ). Thus, exposure to the RyR channel activators and inhibitors identified here could trigger adverse contractile responses in muscle cells and affect proper brain development, especially in susceptible individuals.

Conclusion
The HTS method described herein allowed the elimination of 98% of the compounds as nega tive hits. Furthermore, we were able to cor rectly identify compounds that were previously shown to inhibit or induce a particular enzymes or receptor; however, we also discovered new effects of some xenobiotics. For example, the inhibition of CES1 and CES2 by triclosan was totally unexpected, as was the inhibition of the RyRs by chloranil and dichlone. These in vitro results raise significant biological/toxi cologic questions and further in vivo studies are necessary before drawing any conclusions on the health risks associated with any of these compounds by these specific mechanisms.
Overall, our study shows the feasibility of using combined HTS assays as an approach toward obtaining toxicologic data on the many thou sands of anthropogenic compounds for which there is little if any information. Furthermore, the HTS assays were very useful for quickly identifying compounds of potential risk for further studies, thus concentrating resources on the potentially most significant chemicals. The National Library of Medicine has developed the infrastructure to screen com pounds on possible pharmacologic leads and to report the data in an easily acces sible publically available format; this is part of the National Institutes of Health Molecular Libraries Roadmap initiative. The results for the screening of sEH in this sys tem are available online (National Center for Biotechnology Information 2009); the AhR CALUX bioassay is currently used in the same program. One useful rapid approach would be for investigators or the National Institute of Environmental Health Sciences to propose toxicologically relevant assays and also pro vide environmentally or industrially important compounds to the system.