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r26-491X ./fM 0M 2 0TJOJ.00/0 C opvncm e 0 T)\# A nxtncan So ciety for Phorm ocoloy u d l i p w All n * n u of reproduction m a n y form ra--ro w l vtO L K C l LA * PHARM ACOLOGY. 3 4 :0S 2 - 6M w t l l Thoropoutie A Competitive Binding Assay for 2,3,7,8-Tetrachlorodibenzo-pDioxin and Related Ligands of the Ah Receptor * t C H R ISTO PH ER A. BR A D FIELO and ALAN POLAND HfpArdla LborMtgp'tor Canear Rasaarcn, Univarsity ot Wisconsin, Madison, Wisconsin 53706 Received May 16. 1988: Accepted August 4. 1988 SU M M ARY *' ' A sensitive competitive binding assay for the detection of 2.3.7,8tetrachiorodib^pzo-p-dioxin (TCDD) and other ligands of the Ah receptor w as developed using a stable preparation of the Ah receptor, the 40-55% ammonium sulfate fraction of liver cytosol from C 5 7 B L/6 J mice, and the radioligand [125ll2-iodo-718-dibromodibenzo-p-dioxin (specific radioactivity. 2176 Ci/mmol. and binding affinity. K0 = 6.5 pm ). Conditions are desenbed which maximize assay precision and sensitivity, while minimizing sam ple counting time, ensunng ligand solubility, and permitting at tainment of binding equilibrium for competing ligands. A ssay conditions were developed to allow calculation of the binding affinity for competing ligands and to ensure that an unknown competitor could be quantified in terms of T C D D binding equiv alents.' Standard assay conditions consisted of incubation of 8 pM radioligand and 18-20 pm Ah receptor with 5-1000 pm TCD D. in a 1-ml volume, for 16 hr at 4 Statistical analysis of the standard curve of bound radioligand versus the log of the con centration of competing TCDD indicated the minimal detectable concentration of TCDD to be 10 pm (3.2 pg in a 1-ml assay a < 0.01). The sim plicity, sensitivity, and reproducibility of this com petitive binding assay should prove useful as a screen to detect planar halogenated aromatic hydrocarbons and other ligands or the A h receptor. The availability of this ,25l-labeled dioxin con gener also permitted the charactenzation of Ah receptor-iiganc binding over a range of ligand and receptor concentrations not possible with currently available 3H-ligands. 2.3.7.8-Tetrachlorcxiibenzo-p-dioxin is one of the most potent small molecule toxins known 11-3) and serves aa the prototype for a large number of planar halogenated aromatic hydrocar bons which elicit their biological effects by a common mecha nism le.g., certain isomers of halogenated dibenzo-p-dioxins, dibenzofurans, azo(xy)benzenes, and biphenyls). The risk pre sented by the widespread dispersion of these compounds into the environment is a function of l) their toxic potency, 2) their resistance to chemical and biological degradation (4), and 3) their lipophilicity and hence potential for accumulation in the food chain (5). Analysis of trace concentrations of these compounds in en vironmental and biological samples has been made possible through recent advances in chromatographic and mass spectral technologies. Current methodologies involve varying degrees of sample preparation, separation of isomers by liquid or gas chromatography, and identification and quantification of con- This work was supported in part by the National Institute of Environmental Health Sciences Grant ES-01884. National Cancer Institute Core Grant-07175, and National Cancer Institute Postdoctoral Training Grant T32-CA09020. geners by mass spectrometry. Extremely low concentrations of TCDD in environmental samples, picogram per gram levels (i.e., parts per trillion), are now routinely quantified using this technology (6). Despite the sensitivity of mass spectrometevbased methods, their use is limited by cost and availability of instrumentation. Bioasaays have played a prominent early role in the identi fication and monitoring of chlorinated dibenzo-p-dioxins: e.g., the formation of pericardial edema in the newborn chick (7), and the production of chloracne in the rabbit pinna (8). More recently, cell culture bioassays (e.g., the induction of aryl hy drocarbon hydroxylase activity in rat hepatoma cells (8). and keratinization in XB/3T3 cell cultures (10) | have achieved remarkable sensitivity, 10 pg of TCDD, but have not gained widespread use. Radioimmunoassays have been developed to detect TCDD, 2,3,7,8-tetrachlorodibenzofuran, and chlorinated biphenyls with detection limits approaching 25 pg (11-13). The limited use of these radioimmunoassays may be attributable to 1) the need to characterize each antisera for its reactivity toward a large number of isomers and cross-reacting com- AB8REVIAT10N 3: TCDO. 2.3.7.8-tetracfilorod*)enzo-p-dioxm: M OPS. 3-<N-morpnoiino)prooane3uiforec aod; EOTA. (etnytenedinitntoHGtixsce'ac aod); K0. equiuooum dissociation constant L , ligand (labeled or untaoeiedh A . receptor. R L . receptor-radioligand complex; R L '. transformed reoicvoi-- radxxigand complex: (L]r. total radioegand concentration; (fl]r . total receptor concentration: concentration of receptor determined by Scaich ^ c analysis: B . bound radioligand at tqu*bnum: So. receptor-bound radioligand m the presence of no competing ligand: S ,, racaptor-pound rsciciig sn c m tha prasanca ot comparing ligand: C . competing ligand; (C V concentration of 'free* competing ligand at eqintibnum: dissociation rata constant: N S8. nonspecific binding (radtokgand bwang m me presence of a 200-1000-fow exeesa of 2.3.7,8-tatracmorodibenzofuran); EC-, E C . EC,, competing itgand concentration wfitcn produces 20%. 50%. or 80% reduction m specific binding, respectively: f, hart-life of dissocaoon. 882 G ENP 011267 784078 784079 Binding Assay for TCDD 68 pounds, 2) difficulties in the solubility of radioligand (or anti gen), and 3) the limited availability of the radioligand*. TCDD and related haiogenated aromatic hydrocarbons are all approximate isostereomera and produce a characteristic detail (18), and 2) th# receptor concentration in this preparation i slightly ennched compared to cytosol, the lipid contamination is re duced, and binding charactartstics are unchanged for over l year whe: stored at -80*. pattern of morphological and biochemical changes. The biolog ical effects elicited by these compounds appear to result from their stereospecific binding to a soluble protein, the Ah recep tor, and the resultant gene expression which is initiated by this ligand-receptor complex (14-17). In support of this model: for chlorinated dibenzo-p-dioxins, the apparent binding affinities (Kn) of congeners for the Ah receptor correspond to their rankordered potencies to produce biological responses (e.g., induc tion of monooxygenase activity, lethality, chloracne. and tumor promotion). Secondly, in mice there is a genetic polymorphism in the Ah locus that determines the receptor. Strains which M athem atical and S ta tistica l Theo ry U aad to O p tim ize A ssa y S en sitivity Equilibrium binding. The binding of a radioligand. L. to it receptor. R, under conditions of equilibrium, can be descnbc by thlaw of mass action, where the equilibrium dissociation constant. Ko, i: equal to the ratio of the constituents: [A] " concentration cf fret receptor (1 concentration of free radioligand: and (/?.] = concen tration of radioligand-receptor complex. Classically, this interaction is represented as: R + L RL i l) express a high or low affinity receptor are more or less sensitive to the effects of agonists, respectively, and a variety of toxic responses le g., thymic involution, porphyna, epidermal hyper keratosis. and teratogenicity) have been shown to segregate with the Ah allele which determines the high affinity receptor (for a review see Ref. 16). Kn mL\ [RL] 12) We have recently examined the binding of the A h receptor from C57BL/6J mouse liver and (i:AI]2-iodo-7,3-dibromo<iibenzo-p-dioxin (18) and found it to be best characterized by the following model: Since these haiogenated aromatic hydrocarbons produce R + L RL sa RL' =z R' + L i3 ) their biological effects by virtue of their binding to the Ah receptor, a competitive binding assay of sample and radiola beled ligand for Ah receptor occupancy would appear to offer a direct and 3imple assay system. As an analytical method, a competitive radioligand-Ah receptor binding assay ha* a num ber of advantages: 1) theoretical foundation based on the law of mass action, 2) elimination of the biological variability inherent in the use of whole animals or cells, and 3) rapid screening not requiring expensive instrumentation. The sensitivity, and hence utility, of Ah receptor-based com petitive binding assays has been limited to date by the low specific activity of available radioligands (e.g., ('H]TCDD, 58 Ci/mmol). We have previously reported the synthesis and where RL' is a distinct form of the ligand-receptor complex, which has a much slower ligand dissociation rate than does RL. Under the binding conditions employed lAh receptor from C57EL/ 6J mice, incubation at 4*. for 16 hr), the transformed receptor-ligand complex. R L ' , is less than 25% of the total bound ligand: and equations 1 and 2 provide a rough approximation of equilibrium binding.' Optimisation of s competitive binding aaasy. The quantity of radioligand bound to receptor. [AL] or B. at a fixed concentration of radioligand and receptor, is progressively decreased by the addition of increasing concentrations of unlabeled competitor, C. which competes for receptor occupation. The concentration of radioligand bound, as a function of unlabeled ligand added, describes a competitive binding curve and can be derived from the following relationship: binding kinetics of a new radioligand of the Ah receptor, [l"I] 2-iodo-7,8-dibromodibenzo-p-dioxin (18). Thi* radioligand pos I V-l r . I If-k/\ _ Vl.r - o (4) sesses a high affinity for the Ah receptor (Kn m 6.5 pM) and a high specific activity (2176 Ci/mmol). W# now report the use of this radioligand in the development of a highly sensitive competitive binding assay for ligand* of the Ah receptor. where [L\r mconcentration of total ligand, and [/?]r concentration of total receptor. A major goal in daveloping a competitive binding assay is to optimize the precision and sensitivity of the assay. This is accomplish^ by adjusting th# concentrations of radioligand [L]r and receptor [fi]r . Rational Bereon and Yalow (20) and Ekina and co-workers (19) independently developed theoretical solutions to calculate optimum reagent concen Our approach to the development of this competitive binding assay consisted of: 1) synthesis of s radioligand of high specific activity and high receptor affinity: 2) characterization of a stable and reproducible preparation of the Ah receptor 3) use of competitive binding theory to approximate optimal conditions; and 4) refinement of the conditions by expenment. trations. Although these solution* differ in their definitions of sensitiv ity and considerations of error, they predict similar optimal conditions. Beraon and Yalow define conditions which yield maximal sensitivity to thoaa which give an initial ratio of bound to total radioligand (BJ (L]r) 0.33 to 0.5, (A]r 0.5 K 0, and [L\r that approaches zero. Ekir.s and co-workers define optimal conditions as those which yield Bo/(L|r Radioligand 0.5; [/?]r 3 1.25 K 0 and [L\r S 2.25 K a, when considering only counting arror, or which approach (/?lr * 0.5 K 0 and (L]r " 0. with The sensitivity of a competitive binding assay is proportional to the (specific activity of the radioligand x lKol1'* (19)- Thus, use of [,iSI]2lodo-T.S-dibromodibenzo-p-dioxin (2176 Ci/mmol, counting efficiency -70%. Kn m 1.7 K0 for TCDD) should increase assay sensitivity approximately 6-fold compared to us# of (1,6*JH)TCDD (58 Ci/mmol, counting efficiency -40%). increasing experimental error. These derivations assume simple mass action and equilibrium conditions which only approximate ligand bind ing to the Ah receptor (i.e., equations 1, 2. and 4). Additionally, they are dependant upon experimental as well as counting errors. Therefciv, we used these theoretical values to determine initial ranges of tvceptcr and radioligand concantrationa and further optimized assay s&as.civicy empirically. Rcaptor Preparation The 40-55% ammonium sulfate precipitate fraction of hepatic cy tosol from C37BL/6J mice was chosen because l) we have recently characterized the kinetics of radioligand binding to this preparation in 1This assumption was necessary since the optimization theory u sai was derived from qa. I. 2. and 4. The assumption was supported by th* fee: the. optimal sensitivity was achieved in accordance with the theoretically c*t*rrr.:.. optimal reagent concentration range*, lese "Results' and 'D iscussion') GENP 011 i m 'f 6 8 4 Sradflaid and Poland Quantification of competing Uganda with differing receptor affinity. A host of hslogenated aromatic hydrocarbon isomers are ligands for the Ah receptor, but differ widely in their binding affinities. We foresee this competitive binding assay as being useful in the quantificstion of the sum of these compounds present in environmental or biological samples, in terms of "TCDD binding equivalents." Ekins et aL (19) and Rodbard and Lewald (21) have examined the complex relationship between the relative binding potency of an unlabeled competitor. C. with an equilibrium dissociation constant Kc. and radi oligand. L (with affinity K t,). When the affinity of the radioligand is equal to or greater than that of the competing ligand (and the ratio of bound radioligand/free radioligand is less than 0.5). the estimate of binding potency is nearly linear (i.e.. EC* /EC*it * K,/Ko). Since we have chosen a radioligand with a receptor affinity approximately onehalf that of TCDD. it is very likely the estimate of binding equivalency of unknown ligands will always be nearly linear. We have chosen to optimize the assay sensitivity for detection of TCDD; one may enhance the sensitivity of detecting a known ligand of lower affinity by adjusting the concentration of receptor (A|r and radioligand [L]r (19). Tim e for equilibrium . To accurately estimate the binding equiv alency of a competing unlabeled ligand, it is important that binding equilibrium be achieved for both the unlabeled compound and the radioligand. The time required to achieve equilibrium binding is a function of the dissociation rate constant of the slower dissociating ligand. Since the radioligand has a very high affinity for the Ah receptor, comparable to or greater than that of any known competing ligand, we assumed that 5 times the half-life of its dissociation (r., - 1.9 hr (18)| or 3.5/..I is a good approximation of equilibrium conditions for ail potential ligands (22). Calculation of dissociation constants. The equilibrium dissocia tion constant of a competing ligand (Kc) is most commonly calculated by the Cheng-Prusoff equation (23), which is applicable to situations where [L\ = (Z.]r- These conditions are not met in competitive binding assays which have been optimized for sensitivity (i.e.. Bo/[L\T 0.30.5). We estimated the K, of competing ligands from EC*> values by the equations of Linden (24) which place no constraints on the fraction of radioligand or competitor bound. First the concentration of ` free' competing ligand is calculated: Kg Ko * [L]t + [R]t/2 then [Cf\ is substituted into the following equation to determine Kc' Materials and Methods Reagents. (''I]2lodo-7,8-dibromodibenzo-p-dioxin (21760/ mmol) was synthesized and purified as described previously (18). TCDD was a generous gift of Dow Chemical Co. (Midland. MI), 2,3,7,8Tetrachiorodibenzofuran and 2,3-dichlorodibenzo-p-dioxin were a gift from Drs. David Firestone and Albert Pohland (Food and Drug Admin istration, Washington. DC). ("CJTCDD (specific activity 114 mCi/ mmol), and 2,3-dibromodibenzo-p-dioxin were synthesized as described (25,26). 3,4.3\4'-Tetrachlorobiphenyletherwaea gift from Dr. Andrew Kende (University of Rochester). Estradiol was purchased from Calbiochem (San Diego, CA). Pregenolone-16a-carbonitrile was purchased from Upjohn Diagnostics (Kalamazoo. MI). Sodium phenobarbital was purchased from Merck Chemical Division (Rahway, NJ). Active char coal. grade PX-21. was a gift from Amoco Research Corp. (Chicago, IL). Bacto-Gelattn was purchased from Difco Laboratories (Detroit, MI). Glycerol was purchased from J. T. Baker (Phillipsburg, NJ). EDTA was purchased from EM Scientific (Cherry Hill. NJ). Testos terone, cortisol. Na-L-thyroxine. dithiothreitol. d-mercapcoethsnol. so dium azide, and MOPS (free acid end sodium salt) were purchased from Sigma Chemical Co. (St. Louis. MO). p-Dioxane Ianhydrous 99+% pure) and dimethyl sulfoxide (anhydrous. 99% pure, stored under N? gas) were purchased from Aldrich Chemical Co. (Milwaukee. WI). Buffers. MN represents the stock buffer which contains 25 mM MOPS and 0.02% sodium azide. pH 7.5 (at 4*). MEN is the stock buffer plus 1 mM EDTA. MdENG and MDENG represent MEN with the addition of 10% (w/v) glycerol, plus the addition of either 10 mM d-mercaptoethanol or 1 mM dithiothreitol (respectively). Receptor preparation. The 40-55% ammonium sulfate precipitate of C57BL/6J mouse hepatic cytosol was prepared as described previ ously (18). Standard aaaay protocol. To a series of 12- x 75-mm borosilicate assay tubes was added 5 ui of dimethyl sulfoxide containing TCDD standards (0. 5. 10. 20. 40, 60, 100, 200. 500, and 1000 fmoil or unknowns. The frozen 40-55% ammonium sulfate precipitate (15 mg of protetn/tube) was dissolved in ice-cold MDENG buffer and diluted to a concentration of 150 ug of protein/ral (equivalent to an Ah receptor concentration of 18-20 fmol/ml). The radioligand in dimethyl sulfoxide was added to the solution of receptor to a concentration of 8.0 fmol/mi (--40,000 dpm/mi. added as a 1 1 stock solution/mi of buffer) before dispensing l ml of the solution to each assay tube (without vortexing). The tubes were then incubated at 4* for 16 hr. The assay was terminated by the addition of a 0.5 ml of suspension of charcoai/gelatin (3%/0.3%. w/v) in MN buffer with vigorous stirring on a vortex mixer for 3 sec. followed by incubation for 10 min at 4'C. The tubes were then centri fuged at 2000 x g for 10 min at 4*. A 1-ml aliquot of the supernatant fraction of each tuba was transferred to 12- x 75-mm polypropylene tubas (VWR Scientific, San Francisco. CA). and the bound radioligand was quantified in a MINAXI Series-5000 gamma scintillation counter (United.Technologies/Packard Instrument Co., Downers Grove. IL). Sample counting times were adjusted to obtain a counting error of 3% (range of 0.1-2.0 min. counting efficiency of 75%). Each sample and standard ware analyzed in quadruplicate. Total radioligand. [L]r. was determined by the transfer of 0.67 mi of assay solution directly to the polypropylene tubes for counting (no charcoai/gelatin is added to these tubee). Data analysis. The y -scintillation counter was connected by a serial communication port to an IBM-XT personal computer equipped with software for analysis of radioimmunoassay data and quality con trol (SECURLA PLUS. Packard Instrument Co.. Downers Grove. IL). Total radioligand. (L]r, was defined as the concentration of radioli gand in solution after the 18-hr incubation. (This value is generally 10% lower than the concentration of radioligand originally added to the tubes, i.e., 8 pM.) NSB was defined as the amount of radioligand bound in the pretence of 8 nM 2,3,7.8-tetrachlorodibenzofuran (1000fold molar excess). The total radioligand bound at each concentration of competing ligand (i.e., 5-1000 fmol of TCDD/ml) minus the NSB wae defined as the specific binding: Bo - specific binding in absence of competing ligand and B, m specific binding at ax* concentration of competing ligand. To generats a standard curve, the response parameter, fl./flo, i.e.. the ratio of specific binding at each concentration of TCDD relative to the specific binding in the abeence of competing ligand, was plotted uerrua tha log of tha concentration of TCDD. For data analysis we choae to fit these results, via an iterative curve-fitting technique, to the four-parameter logistic model described by DeLean tt aL (27).1The concentration of ligand in unkown samples is quantified ea `equiva lents* of TCDD (in picomolar concentration or ferntomoles per milli liter), by interpolation of the fractional bound response metameter on 1BjBo - Bo - NSB NSB l - <[C1/EC)' here 5 is slope of log logit plot. GEN? 011269 784080 the standard curve. Outliers wart defined aa values varying more than 5 standard deviations from the mean In 4 at each point). The variation between assays was monitored by following a variety of quality control parameters for the standard curve, which included: l) B... (specifically bound ligand in the absence of competing ligand: 2) B../Lr. (specifically bound ligand aa a fraction of total radioligand per milliliter); 3) MSB: 4) estimates of EC.-.., EC.,, and EC*> (competing ligand concentration which produces a 20%. 30% and 80% reduction in specific binding, respectively:), 5) the calculated slope of the fourparameter logistic model (i.e.. slope of the logit-log plot), and 6) a control standard yielding a TCDD concentration of 45 pM. Radioliqond- 2 .4 pM *T * aOn2t (Unding A i u y for TCOO 6 8 5 7 .7 pM 2 3 pM Results A representative saturation binding isotherm of (l*,I]2-iodo7,8-dibromodibenzo-p-dioxin binding to the Ah receptor in the 40-55% ammonium sulfate precipitate fraction of liver cytosol is shown in Fig. 1A. The data in Fig. IA were transformed by the method of Scatchard (28) to generate the plot described in Fig. IB. From an average of three experiments the concentra tion of binding sites, = 120 7 fmol/mg of protein, the apparent Kn 16 3 pM, and the correlation coefficient 0.99 0.00. The Hill coefficient of these plots is 0.97 0.02 with a correlation coefficient =0.99 0.00 (29). As outlined under "Rationale," the approximate conditions for optimal precision and sensitivity of a competitive binding assay are: l) B ../[L ]r * 0.33-0.5; 2) [/? ]r S 1.25 K ,, and [ L ] T 3 2.25 K,,. In Fig. 2, are displayed a senes of competitive binding curves generated at three radioligand concentrations ((LJr * 2.4, 7.7, and 23 pM) and three receptor concentration ([fi]r * 7, 28. and 112 pM), with TCDD as the competing unlabeled ligand. The initial B,,/[L\T ratios (in the absence of unlabeled ligand) varied from 0.2 to 0.7. For a given radioligand concen tration. decreasing the concentration of the receptor decreased the initial B,J[L\t and increased the initial slope of the com petitive binding assay. As a test of the relative assay sensitivity obtained at differing Fig. 2. Effect of radioligand and receptor concentration on binding curves generated with TCOO as competing ligand. A ssays were mcuoateo at 4* for a penod of 16 hr (see text). AD data points are the result of quadruplicate determ inations. Ordinate: total radioligand bound divided by total radioligand in solution. A b scissa: concentration of uniabeteo competitor (TCOO. ptcomolar concentration). Receptor concentration: 112 pm ( ): 28 pm (O ); and 7 pm ). Left, total radioligand concentration. [L ]r. is 2.4 pm (11.600 dpm/mi); center, total radioligand concentration is 7.7 pM (37.200 dpm/ml); right, total radioligand concentration is 23 pm (111.000 dpm/mi). TABLE 1 Statistical power of the competitive binding assay Statistical oowor is footnote 3) of various assay conditions to detect tne decrease m radioligand sound m the presence of 5 pm TCOO. as compared to ramodgana pound m me aosence of TCOO (i.e.. 30). Ekns et a/. (19) have snown mat me sensitivity of a comoetrttve Binding assay is equivalent to tne error m 8 0 divided By me irutiai siooe of tne assay curve, a Bound/ a competing ligand added. The statistics power (1 - cf) was calculated using tne Student s t value witn a s 0 01 (30. 31). Baooegand Concentration Sia t e e Power s i flecsotor Concentration T om 28 ou 1120M P* 2 .4 0.99 0.10 0.00 7 7 0.95 0.30 0.00 23 0 .6 0 0 .1 0 0.10 Binding Isotherm Scatchard Analysis Total Radioligand (fm o l/m l) Total 0ound(fm ol/m l) Fig. 1. A. Equilibrium binding of ('" 112-iodo-7,8-<Jibromodibanzo-p-dioxin to the 40-55% ammonium sulfate precipitate fraction of hepatic cytosol. The receptor preparation (145 *g of protem/mt) w as incuOated at 4* for 16 hr. with increasing concentrations of radioNgand (3-70 pet). Nonspe cific binding was estimsted in the presence of a 200-fotd excess of 2.3.7.8-tetrachtorod>&enzofuran. Each posit represents me average of two determnaoons. 9. Scatcnard analysis of equMPrtum landing data from A. The ending param eters were catcutated By a linear least squares estimate of soeoflcaity bound/free radioligand versus speafleatfy bound radioligand. The K0 ana 8mm results (inset) are derived from the experi ment shown, me mean and standard deviations from three experiments are K0 16 i 3 pm and Bmm 18 x 3 fmol/ml (120 17 fmol/mg of protein). reagent concentrations, we compared the statistical power* of these assay conditions to detect a 5 pMconcentration of TCDD. As shown in Table 1, the power of the assay increased with a reduction of receptor concentration, at a receptor concentration of 7 pM, the power increased as radioligand concentration decreased. TCDD and related halogenated aromatic hydrocarbon have very limited solubilities in aqueous solution. We examined the concentration of radioligand that remained in solution, after a 16-hr incubation at 4*. as a function of the concentration of the receptor preparation used. As seen in Fig. 3. only at protein concentrations of 150 ng/mi ((/?]r 18 pM) or greater, did at least 95% of the added radioligand remain in solution. While maximal sensitivity is achieved at receptor concentrations of 7 pM, we chose to use the higher concentration (18 pM, 150 ug/ ml) to increase the solubility of competing ligands. Based on the observations presented above, the standard conditions for the competitive binding assays were chosen to be: radioligand concentration, [L]r, of 7.2-7.7 pM (*4 x 104 dpm/ml added initially), a receptor concentration of IS--20 phi 1The statistical power of an aaeey (30) ta the probability that the deerv.-^ in bound radioligand, due to the presence of 3 pM TCDD. will be detected .hen compared to B* (Student'! ( value, a s 0.01). Thus. aaaay conditions which yield the greatest statistical power are the conditions which yield the greatest assay sensitivity. Statistical power is equal to l - d (where d is the probability of making a type II statistical error) (31). GENP 011270 784081 % 686 Bmdftatd and Poland COMPETITIVE BINDING CU R V E Fig. 3. Effect of protein concentration on radioligand solubility. The radioligand (8.3 pM) was incubated at 4 with varying concentrations of the receptor preparation (0. 5. 10. 19. 38. 75, 150, 300, 600. and 1200 ng of protetn/ml. After incubation for 16 hr, total radioligand in solution, w as determined as descnbed in the text. Each point represents the average of two determinations. (approximately 150 ug of protein/ml), and an incubation time of 16 hr at 4*. A typical standard curve for the competitive binding of TCDD determined under these conditions is shown in Fig. 4. The initial total bound ligand is 17,176, NSB is 1996 dpm, initial specifically bound ligand. Bo, is 15,180 dpm and BJ[L\t =0.40. The concentration of TCDD which produces a reduction in specifically bound radioligand equal to one*half the initial value (EC.w) is 41 to 3 pM (Table 2). Under the standard assay conditions the minimal detectable concentra tion of TCDD which reproducibly produced a statistically sig nificant reduction in bound radioligand Is 10 pM (10 fmol/ml =3.2 pg/ml, a s 0.05). Thus, to ensure ligand solubility with an increase in [Hr], we have reduced sensitivity about 2-fold. By reducing the assay volume to 0.25 ml, one can decrease the minimal detectable concentration to 0.8 pg of TCDD (data not shown). The advantage of the 1.0-mi incubation volume is greater bound radioactivity, and thus reduced counting time per sample to achieve the same precision. We have previously found that [123I]2-iodo-73*dibromodibenzo-p-dioxin has a biphasic dissociation from the Ah recep tor, with the major component (75%) having a dissociation rat* constant, Jk_, 0.36/hr at 4* ( 7 \ 1,9 hr-1) (18). Thus, binding equilibrium for competing ligands with dissociation rats con stants equal to or greater than that of the radioligand should be achieved by 5 x t.,, or 10 hr (22) (see Rationale). Competing ligands with slower dissociation rates than the radioligand [presumably TCDD, since its K0 is approximately one-half that of the radioligand (Table 3)] may require more than 10 hr to reach binding equilibrium. To confirm that binding equilibrium was obtained for TCDD by 16 hr, we examined the competitive binding curves at 4* os a function of incubation time (Fig. 5). The initial bound radioligand Bo and slope of the competitive binding curved reached a matimum by 16 hr, remained virtually unchanged between 16 and 48 hr, and then progressively de clined. The decrease in Bo at longer incubation times is probably attributable to degradation of receptor with time. The accuracy of the assay is dependent on the stsndszd, i.e,, the accuracy in weighing and dilution of small amounts of TCDD. We checked this by determining the competitive bind- TCDD (pM) Fig. 4. Competitive binding assay. Standard curve with TCOO as com peting ligand. Assay w as run, as descnbed in text, with increasing concentrations of TCDD, a total radioligand concentration of 7.2 ck; (added 8.2 pm), and a receptor concentration of 18 pM. Incubation w as tor 16 fir at 4*. Ordinate: S,/0<>, specifically bound radioligand in the presence of a given concentration of TCDO divided by specifically bound radioligand in me absence of TCDD. Abscissa: concentration of TCDD (ptcomoiar concentration). The first data point is the interpolated concen tration of So, as calculated by the four parameter logistic mode- (see text). For this standard curve; E C - 13 pM, EC m m 40 pM, E C '= 138 pM, So divided by total radkMgand - 0.36. N SS divided by total radioli gand - 0.06. 784082 TA B LE2 QusMy control partmetera of me competitive binding seeay standard curve The standard curve tor TCOO compaction with radioligand tor theAh receptor was detarmined aa daacnbad under `Matanats and Methods.' Quality cortrot parametws ware compared from assays using tresnty prepared radioligand (n - 9). and usmg radioligand aftar 75% radiodecay (120 days, n - 1). Values are means = standard deviation. Al parametersare descnbed in me text. NO. not detemwied. tam ar He*' Ratidgand NoRaeodKay Raaotgrd afte 75% Raaedusy So/Lr HSBfLr ECn(pM ) ECm (PM) ECm (pM) Logit slope 45 pm TCOO (control pool) 40 pM ('"CITCDO 0.36 to 0.06 0.06 s; 0.01 14 to 3 41 to 3 140 to 20 1.3 0.1 42 6 1 40 2 0.34 0.06 14 45 160 1.2 46 NO GENP 011271 784083 Binding Assay for TCOO 687 TA8LE3 Relative binding potancias for Uganda of the Ah racaptor The aynostitrvs wnomg assay was parformaa usmg nonraOtoiaoatad conganars. under standard conations as dascnoad unoar 'Matenats and Metnods ' The (R), was 20 pm and |L|r - 7-7 6 pm. The true Ka of me racnongana was assumed to De 6 5 pm 118) The EC, '* tna total ligand concentration wnicn produced a 50% reduction m the specific omoing of tna radioligand: tna relative omding potency is c r ia . n ---- The Kc lor eacn comoound was determined Oy tna metrod of Undan (Eqs. 5 and 6. Ref 2 0 and tne relative * c " *c compantor/Ke TCOO. ComotWW EC Kn m EC aM0v 2 .3 .7 .8-Tetracniorodibenzo-p-O ioxin 2-1000-7.8-O ibrom ooibenzo-p-dioxn 2 .3 .7 .8 - T etracntorooibenzofuran 2 .3-Oibrom ooibenzo-p-Oioxin 2 .3-0icW oro0i0enzo-p-dioxin 9" 41 58 85 580 6400 1 1.4 2.1 14 160 9* 68 11 18 140 1600 1 1.7 2 .8 20 230 benzo-p-dioxin in the radioligand stock solution. The K,, of the 2,3-dibromodibenzo-p-dioxm was estimated by competitive binding to be approximately 140 pM (see below). We tested whether radioligand decay products might compete for receptor binding and alter the competitive binding curve. Assays using the stock solution of radioligand 120 days after its synthesis (75% of '*'1 radiodecay), showed no significant deviation in the quality control parameters compared to freshly prepared radi oligand (Table 2). Thus, repunfication of the radioligand by high performance liquid chromatography does not appear nec essary for at least 120 days after synthesis. Using the standard assay conditions outlined above, compet itive binding curves were generated for a variety of halogenateddioxin congeners (examples are given in Fig. 6). The relative binding affinities of these congeners, as indicated by estimates of EC*, and Kn (Table 3), are in agreement with relative binding affinities, and biological potencies which have been published previously (i.e., TCDD > TCDBF > 2.3-dichlorodibenzo-pdioxin > 3,4,3\4'-tetrachlorobiphenylether (10, 25)|. Addition ally, testosterone, phnobarbital, thyroxine, estradiol, cortisol, and pregnenalone-l6a-carbonitnle at levels as high as O.S uM (i.e., 10s times the concentration of radioligand) did not com pete with the radioligand for Ah receptor occupancy (data not shown). D iscussion In this report, we describe a sensitive competitive binding assay for the detection and characterization of ligands of the Ah receptor. Since the sensitivity of such an assay is a function of the binding affinity and specific activity of the radioligand, considerable attention has been devoted to the selection, label Fig . 5. Effect of mcuoation time on the comoetrtion binding curve. The assay w as run as described in the text, with increasing concentrations of TCO O. a total radioligand concentration of 7 5 pm and a receotor concentration of 18 p m . The com petition binding curves w ere analyzed after varying incubation times (key is mset) at 4* Ordinate: radioligand ing, and binding kinetics of this compound. [u'I]2-iodo-7,8dibromodibenzo-p-dioxin (18). The higher specific acitivity of this radioligand (2176 Ci/ mmol) and the greater counting efficiency of y -emission ovei- specifically bound m disintegrations per muMiter. A b scissa: concentration of uniaoeted TCOO (picom oiar concentration). Each value is tne result of tnpiicate oetermmations. d-emission increases the ratio of counts per minute per femtomole of radioligand about 2 orders of magnitude as compared to [`H1TCDD (58 Cl/mmoi). The 1A'I-labeied ligand offers other minor advantages: 1) it has high radiochemical punty resulting from the easy separation of the iodinated product from the starting material, reaction intermediates, and radiodecay prod ucts, 2) specific activity is determined as that of the Na(u!,l] used for synthesis, and 3) after 75% radiodecay, a stock solution - o , : f the '^I-ligand produces the same competitive binding curve, * o a indicating that radiodecay products do not significantly affect receptor-ligand binding (Table 2). In a previous report (18), we described the effects of protein concentration on the quantification of "free" radioligand and on the calculations of K0 and (RJr. We suggested that at protein 0 Concentration of C o m p atilo r (M ) concentrations above 70 ng/mi "nonspecifically bound" radi Fig. 6. Competition by vanous compounds for tne specific binding of oligand is misclassified as "free" radioligand, leading to signif "d ['" l]2Hod<>7.8-dit)romodibenzo-p-dioxin to the Ah receptor. The assay icant overestimation of Ko by Scatchard analysis. By minimz- conditions are identical to those descnped in Fig. 5. A l values are the result of quadruplicate determination*. Compounds were dissolved in ing misclassification errors we calculated the Kn of (`-"I^-iooo- dimethyl sulfoxide and addad to tne ncudation m 5 total volume. 7,8-dibromodibenzo-p-dioxin to be approximately 6.5 pkc. Fei- the competitive binding assay, a concentration of 150 ug/ml 3 ing of [l4C]TCDD. Three independent analyses of a ("C]TCDD waa employed to ensure ligand solubility. At this protein con standard, quantified by d-scintillation spectrometry to a con* centration Scatchard analysis of the saturation binding isoth O centration of 40 pM, gave a vaiua of 40 2 pM from the erm yielded an apparent K0 of 16 pM (Fig. 1). suggesting that standard curve (Table 2). some misclassification of nonspecifically bound radioligand as o '*'1 decays to tellurium with a half-life of 60 days. Radiodecay free radioligand ia occurring. The effect of this misclassification of the iodinated ligand may produce unlabeled 2,3-dibromodi* appears to be minimal, as the calculation of Kc for 2-ioec-7,- dibroroodibenzo-p-dioxin (Le., 11 pm) is only 1.7-fold greater than the previously determined estimate of K0 for the l4,I analogue (i.e., 6.5 pM). This 1.7-fold overestimation of Kc suggests that the true Kc for TCDD is approximately 4 pM, 100-fold lower than previous estimates for this compound (25). It has been previously proposed (32) and demonstrated (33) that a competitive binding assay, using the Ah receptor and radioligand, could be used to screen for the presence of com peting ligands present in the environment. The extraordinary sensitivity of the present assay, attributable to the high specific activity of the radioligand, revised estimate of receptor affinity, and optimization of assay sensitivity, make it feasible to screen environmental samples for TCDD and related halogenated aromatic hydrocarbons: e.g., chlorinated dibenzo-p-dioxin, -dibenzofuran, and -biphenyl isomers. Environmental samples may also contain polycyclic aromatic hydrocarbons, many of which are also ligands of the Ah receptor. These compounds can be eliminated from analysis by the appropriate sample cleanup. The method of sample cleanup depends on the sample matrix te.g., biological tissue, soil, water, etc.) and the presence of interfering substances. The competitive binding assay does not provide chemical identification of competing ligands, but an estimate of their concentrations as "TCDD-binding equiv alents'' (see Rationale and Table 3). Samples judged to have sufficient concentrations of TCDD-binding equivalents, could be subjected to mass spectrometry for chemical identification. Finally, one of the most exciting uses of this competitive binding assay, is to screen biological tissue extracts for the postulated endogenous ligand of the Ah receptor. References t. Poland. A., and A. Kende. 2.3.7.8-Tetrachlorodibenzo-p-dioxin: environmen tal contaminant and molecular proba. Fed Proc. 35:2404-2411 11976). 2. Schwetz. B. A.. I. M. Norm. G. L Sparachu. V. K. Rowe. P. J. G ehnnf. J. L. Emerson, and C G. Gerbig. Toxicology of chlorinated dibenzo-p-dioxins. Em iron Health Penpect 5:87-99 11973). I Kuciba. R. I . D G Keyes. J. E. Beyer. R. M. Carreon. C. E. Wade. D. A. Dittenoer. R. P Kalnina. L. E. Frauaon. C. N. Park. S. D. Barnard. R. A. Hummel, and C G. Humiston. Results of a two-year chronic toucity and oncogenicity jtudv of 2.3.7.3-tetracnlorodibenzo-p-dioxin m rata. Toxicol A ppl Pharmacol 48:279-303 11978). a Kearney. P C.. E. A. Woolaon. A. R. Isenaoe. and C. S. Helling. Tetrachlorodtbenzodiozin in the environment: source*, fate, and decontamination. Environ. Health Penpect. 5:273-277 (1973). V Bickei. M. H . and S. Muehlbach. Pharmacokinetics and ecodisposition of polyhalogenated hydrocarbon*: aapecta and concept*. Drug Metad. Rev 11:149-190(19801. 6. Smith. L M.. D. L Stalling, and J. L Johnson. Determination of part-pertrillion levels of polych Ion noted dibenzofuran* and dioxin* in environmental samples. A nal Chem. 56:1830-1842 (1984). 7. Higginbotham. G. R.. A. Huang, D. Firestone. J. V erm , J. Reea. and A. D. Campbell. Chemical and toxicological evaluations of iaolatad and synthetic cnloro derivatives of dibenzo-p-dioxin. N ature ( L a n d ) 220:702-703 (1968). 8. Jones. E. L . and H. Knxek. A technic for testing acnegemc potency m rabbits, applied to the potent acnegen. 2J.7.3-tetrach!orodibenxo-p-dioxin. J Invest. Dermatol 3*511-5 1 7 (1 9 6 2 ). 9 Bradlaw. J A . and J. L Caateriine. Induction of enzyme activity in cell culture: a rapid screen for detection of planar polychlorinated organic com pounds. J Assoc Offic. A n a l Chem. 62:904-916, (1979). 10. Knutson. J C . and A. Poland. Kermtinization of mouaa teratoma cell line XB produced bv 2.3.7.8-tetrachlorodibenzo-p-dioxin: an in vitro model of toucity. Cell 22:27-36 1 1980). 11. Albro. P. W . M. I. Luster. K. Chae. S. K. Chaudharv. G. Clark. L. D. Lawson J. T. Corbett, end J. D. McKinney. A radioimmunoassay for chlonnatec dibenzo p-dioxina. Toxicol A ppl Pharmacol 50:137-146 11979). 12. Luatar. M. I.. P- W. Albro. K. Chae. L D. Lawson. J. T Corbett, and J. D McKinney. Radioimmunoassay for quantitation of 2.3.7.3-tetrachlordiben tofuran. A nal Chem. 52:1497-1500(1980). 13. Luatar. M. I.. P- W. Albro. G. Clark. K. Chae. S. K. Chaudhary. L. D Lawson J. T. Corbett, and J. D. McKinney. Production and characttnzation oi antisera specific for chlorinated biphenyl species: initiation of s radio immunoassay for aroclors. Toxicol A ppl Pharmacol 50:147-155 U97S). 14. Goldstein. J. A. Structure-activity relationships for the biochemical effects and the relationships to toxicity, in Halogenated Biphenyls. Terphenyls Naphthalenes, Dibenxodtoxins and Related Products iR. Kimbrough, ed.l Elsevier/North-Holland Biomedical Press. Amsterdam 1 1960). 15. Poland. A.. W. F. Greenlee, and A. S. Ksnda. Studies on the mechtnism ot action of tha chlorinated dibenzo-p-dioxina and related compounds. Ann. .V> .4cod. S et 320:214-230 11979). 16. Poland. A., and J. C. Knutson. 2.3.7.8-Tetrachlorodibenzo-p-dioxm and relatad halogenated aromatic hydrocarbons: examination of tha mechanism oi toxicity. Annu. Rev. Pharmacol 22:517-554 11982) 17. Jones. P B. C.. L K. Dumn. D. R. Gaieazzi. and J. P Whitlock. Control or cytochrome P.-450 gene expression: Analysis of a dioxin-responsive enhancer system. Proc. N a tl Acad. Scl USA 83:2802-2806 11986 18. Bradfield. C. A.. A. S. Kende. and A. Poland. Kinetic and equilibrium studies of Ah receptor-ligand binding: use of (1" !]2 iodo-7.8-dibromodibenxo-pdiozin. M ol Pharmacol 34:229-237 11988). 19. Ekins. R. P.. G. B. Newman, and J. L. H. O'Riordan. Theoretical aspects ot "saturation* and radioimmunoassay, in Radioisotopes in Medicine: fn Vitro Studies iR. L. Goswtiz and B. E. P Murphy, eds.). U S . Atomic Energy Commission. Oak Ridge. TN 11968). 20. Berson. S. A., and R. S. Yalow. Quantitative aspects of the reaction between insulin and insulin binding antibody J Clin. Invest 38:1996-2016 1959* 21. Rodbard. D.. and J. E. Lawaid. Computer analysis of radioligand assay and radioimmunoassay data. Acta Endocrinol 64!suppt. U7):79-103 (1970 22. Motulaky. H. J.. and L C. Mahan. The kinetics of competitive radioligand binding predicted by the lew of mass action. M ol Pharmacol 25:1-9 119641. 23. Chang, Y.-C.. and W. H. Pruaoff. Relationship between the inhibition con stant (Ki) and the concentration of inhibitor which causes 50 percent inhi bition l(M) of an enzymatic reaction. Btochem. Pharmacol 22: 3099-3106 (1973). 24. Linden. J. Calculating the dissociation constant of an uniabeied compounc from the concentration required to displace radiolabel binding bv 50^ J Cyclic. NucL Res. 8:163-172 11982). 25. Poland, A.. E. Glover, and A. S. Kanda. Stereospecific, high affinity binding of 2,3,7.8-tetrachlordibenzo-p-dioxin by hepatic cytosol. J Biol Chem. 251:4936-4946 11976). 26. Kande. A. S.. J. J. Wade. D. Ridge, and A. Poland. Synthesis and Founer transform carbon-13 nuclear magnatic resonance spectroscopy of new toxic potyhaiodibenxo-p-dioxina. J Org. Chem. 39:931-937 (1974). 27. DeLean, A.. P. J. Munson, and D. Rodbard. Simultaneous analysts of families of sigmoidal curve*: application to bioaasay. radioligand assay, and physio logical do*e-response curve*. Am. J Physiol 235:E97-E102 I197S). 28. Scatchard. G. The attractions of protains for small molecules and tons. Ann. N Y A e a d S e t 51:666-671, (1949). 29. Ben net, J. P., and H. 1. Yamamura. Neurotransmitter, hormone-, or drug receptor binding methods, in Neurotransmitter Receptor Binding lYamamurm. H. 1.. Enna. S. J. and Kuhar. M. J.. ada.). Raven Press. New York (1985). 30. Owen, D. B. The power of Student's t-test. Am. Statist. Assoc. J 60:320-333 11965). 31. Box. G. E. P.. W. G. Hunter, and J. S. Hunter. Statistics for Experimenters. John Wiley A Son*. Inc.. New York (1978). 32. Poland. A- and E. Glover. 2.3.7.8-Tetrechlorodibenzo-para-dioxin and en zyme induction, in Chlorinated Phenory Acids and Their Dioxins tC. Ramei. ed.). Ecological Bulletins, Swedish Natural Science Reeearch Council. Berlingx, Lund. Sweden 11978). 33. Toftgard. R.. G. Lofroth. J. Carlatedt-Duke. R. Kurt, and J.-A. Gusursaon. Compounds in urban air compete with 2.3.7,8-utrachlorodibenxo-p-dioxin for binding to the receptor protein. Chem.-Biol Interact. 46:335-346 11SS3). Send rep rin t requeete to: Dr. Alan Poland. McArdle Laboratory for Cancer Reeearch. University of Wisconsin. Madison. W! 53706. G E N P 0 1 1273 784084