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The examination and quantitation of tissue cytosolic receptors for 2,3,7,8-tetrachlorodibenzo-p-dioxin using hydroxylapatite
ANALYTICAL
BIOCHEMISTRY
124,
I- 11 ( 1982)
The Examination and Quantitation of Tissue Cytosolic Receptors 2,3,7,8-Tetrachlorodibenzo-pdioxin using Hydroxylapatite T. A. GASIEWICZ’
for
AND R. A. NEAL*-’
Department of Radiation Biology and Biophysics, The University of Rochester, Rochester, New York 14642, and *Department of Biochemistry, Vanderbilt University, Nashville. Tennessee 37232 Received November 20, 1981 An assay using hydroxylapatite has been developed for the examination and quantitation of cytosolic receptors for 2,3,7,8-tetrachlor~ibenzo”~-dioxin (TCDD). This method, as compared to others, has a relatively high ratio of specific to nonspecific binding of [3H]TCDD and is relatively rapid. The total number of specific binding sites and equilibrium dissociation constants (Kn) for [‘H]TCDD were determined in hepatic cytosol from Sprague-Dawley rats and C57BL/6J, DBA/ZJ, and B6D2FI/J mice. With the exception of the cytosol from DBA/ 25 mice in which little specific binding was observed, high-affinity, specific binding was obtained in ail cases. The rat hepatic cytosol demonstrated the highest affinity for [‘H]TCDD with a Kn of 0.12 nM. The total amount of specific [3H]TCDD binding in hepatic cytosol from B6D2F,/J mice was found to be intermediate between that of the C57BL/6J and DBA/ZJ parents. Ligand competition studies suggest the specificity of binding of various compounds to the receptor species from Sprague-Dawley rats is similar to that observed with the hepatic cytosol from C57BL/6J mice. These data support the hypothesis that, as in mice, the particular cytosol binding species in rats is the receptor that may mediate the induction of aryl hydrocarbon hydroxylase activity.
TCDD3 is formed as a contaminant in the synthesis of 2,4,5-trichlorophenol and is considered on both an acute and chronic basis to be one of the most toxic synthetic compounds known (1). This compound is a representative of a group of structurally related halogenated aromatic hydr~arbons ineluding other dibenzo-p-dioxins, biphenyls, dibenzofurans, as well as the azo- and azoxybenzenes, all of which produce similar patterns of toxicity and bi~hemi~al responses (2-5). The mechanism(s) of action of these compounds is unknown.
TCDD has been shown to be a potent inducer of two or more forms of cytochrome P-450 and approximately 20 associated monooxygenase activities (including aryl hydrocarbon hydroxylase (AHH) (6,7), as well as microsomal UDP-glucuronosyltransferase (S), cytosolie reducedNAD(P):menadione oxidoreductase (91, and ornithine decarboxylase (10). In certain strains of mice, the induction of these enzymes is regulated by a single genetic locus, the Ah locus ( 11-l 3). The stereo-specific, high-affinity binding of TCDD and its congeners to a cytosolic receptor protein appears to correlate with the ability of these compounds to induce AHH activity (responsiveness) (14), as well as elicit toxic effects in various animal species (2,15). The translocation of this TCDD-receptor complex to the nucleus, subsequent nuclear binding, and alteration of gene expression appear to be essential events in the TCDD-mediated in-
’ To whom requests for reprints should be addressed. * Present address: Chemical Industry Instituteof Toxicology, P.O. Box 12137, Research Triangle Park, N. C. 27709. 3 Abbreviations used: TCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxin; AHH, aryl hydr~arbon hydroxylase; HAP, hydeoxylapatite; TCDF, 2,3,7,8-tetrachlorodibenzofuran; Hepes, 4-( 2-hydroxyethyl)- 1-piperazineethanesulfonic acid, MOPS, morpholinopropanesulfonic acid. 1
0003-2697/82/l
10001-l 1$02.00/O
Copyright 0 1982 by Academic Press, Inc. All rights of reproduction in any form reserved.
2
GASIEWICZ
duction of AHH activity (16). Aromatic hydrocarbon hydroxylase activity regulated by the Ah locus appears to be a significant step in the metabolism of certain polycyclic aromatic hydrocarbons (6) and may alter their ability to induce carcinogenesis in various strains of mice (17,18). TCDD has been reported to be a potent carcinogen in chronic feeding studies using rats and mice (19-21). However, the covalent binding of TCDD and/or its metabolites to tissue macromolecules was found to be 4 to 6 orders of magnitude lower than most chemical carcinogens (22). Recent studies suggest TCDD is not mutagenic in bacterial systems (23-25). Studies by Pitot et al. (26) indicate that TCDD is an extremely potent promoting agent. It has been proposed that its action as a promoter may be mediated by its binding to a receptor and the subsequent alteration in gene expression (26). Previous investigators have utilized dextran-charcoal adsorption techniques (14), isoelectric focusing (27) and sucrose-density gradients (28,29) for the assay and examination of TCDD cytosolic receptors. These methods possess a number of disadvantages including a high level of nonspecific binding of [ 3H]TCDD, inconsistency, possible alteration of the receptor by trypsinization, as well as being time consuming. In this report, a method is described for the quantitation of the cytosolic receptor for TCDD utilizing hydroxylapatite (HAP). Similar procedures using HAP have been applied to the study of steroid receptors (30,31). We have found this method to be specific and relatively rapid for the study of TCDD receptors. Additional properties of these receptors in rats and mice are also described. MATERIALS
AND METHODS
Materials. [ 1,6-3H]TCDD (50.5 mmol) was synthesized and purified as viously described (32). The compound >98% chemically pure as determined
Ci/ prewas by
AND NEAL
high-performance liquid chromatography (33). TCDF was a generous gift of Dr. J. A. Moore (NIEHS, Research Triangle Park, N. C.). Benzo[cu Jpyrene, 3-methylcholanthrene, Triton X- 100, polyethylene sorbitan monooleate (Tween 80), dithiothreitol, Hepes, and MOPS were purchased from Sigma Chemical Company (St. Louis, MO.). BNaphthoflavone was purchased from Aldrich Chemical Company (Milwaukee, Wis.). 1,2,4-Trichlorodibenzo-p-dioxin and dibenzo-p-dioxin were obtained from Analabs (North Haven, Conn.). 1,2,3,4,7,8Hexachlorodibenzo-p-dioxin was purchased from KOR Isotopes (Cambridge, Mass.). DNA-grade HAP was purchased from BioRad Laboratories (Rockville Centre, N. Y.). Decolorizing charcoal (Neutral Norit A) was obtained from Fisher Scientific (Rochester, N. Y.). Liquescint scintillation cocktail was purchased from National Diagnostics (Somerville, N. Y.). Animals. Male Sprague-Dawley rats (200-250 g) were purchased from Charles River (Wilmington, Mass.). Male C57BL/ 65, DBA/2J, and B6D2F1/J mice (20-22 g) were obtained from Jackson Laboratories (Bar Harbor, Me.). Animals were housed under laboratory conditions for at least 3 days prior to use. They were fed commercial rat or mouse chow (Ralston Purina, Richmond, Ind.) and water ad libitum. Tissue preparation. Animals were killed by decapitation or cervical dislocation and their livers were excised and weighed. These tissues were homogenized in 3 vol of HEDG buffer (25 mM Hepes, pH 7.4; 1.5 mM EDTA; 1 mM dithiothreitol; and 10% glycerol (v/v)) in a glass homogenizer fitted with a Teflon pestle. For mice, 2 or 3 livers were pooled for a single preparation. Unless specified, these and the remaining procedures were carried out at 0-4°C. Each homogenate was transferred to polycarbonate tubes and centrifuged at 10,OOOg for 20 min. The supernatant fraction was collected and centrifuged at 105,OOOg for 60 min. Following each centrifugation the turbid lipid layer was
ASSAY
FOR
2,3,7,8-TETRACHLORODIBENZO-p-DIOXIN
removed with a Pasteur pipet. Following the last centrifugation the cytosol was removed, taking special care not to disturb the microsomal pellet. The protein concentrations of the hepatic cytosol preparations were estimated by the absorbance at 215/225 nm (34) and later confirmed by the method of Lowry er al. (35) using bovine serum albumin as a standard. The protein concentration was diluted with HEDG buffer to between 1.5 and 2.5 mg/ml. The diluted cytosol was used within 1 h after preparation. HAP standard aiming assay. DNA-grade HAP was washed with HEDG buffer, pH 7.4, until the pH of the washes remained at 7.4. The HAP was then resuspended in 2 vol of HEDG buffer at 0-4°C with gentle shaking. The incubation conditions for this assay are similar to that previously described by Poland et al. (14). Two milliliters of the 105,OOOg supernatant fraction with a protein concentration of 1.5 to 2.5 mg/ml was incubated with various concentrations of [3H]TCDD (0.1-1.0 nM) and with [3H]TCDD plus a 200-fold excess of unlabeled TCDF for 2 h at 20°C in a shaker water bath. The [ 3H JTCDD and [ 3H]TCDD + TCDF were added to the incubations in a volume of 5 ~1 p-dioxanelml of cytosol. After 2 h, 0.2 ml of the incubation was removed and added to a scintillation vial, and the total [3H]TCDD contained per volume of incubation mixture was determined. An additional 0.2 ml of this mixture was also removed and added to a disposable 13 X 1OOmm glass test tube containing 0.25 ml of the HAP suspension. These were incubated for 30 min at 0-4°C with gentle shaking every 10 min. At the end of this time, 1.0 ml of ice-cold HEDG buffer containing 0.5% (v/ v) of Tween 80 was added, These tubes were shaken in a Vortex mixer and centrifuged at 1500 rpm for 5 min. The HAP pellet to which the cytosolic protein had been adsorbed was washed three additional times with 1.0 ml of the HEDG buffer containing
RECEPTORS
3
Tween 80. Following each centrifugation the supernatant was drawn off using a Pasteur pipet. After the last wash, 1.O ml of absolute ethanol was added to the tube containing the HAP pellet. The contents were mixed using a Vortex mixer, removed with a Pasteur pipet, and added to scintillation vials. The tubes and the pipets were washed with an additional 1.0 ml of absolute ethanol, and this wash was also added to the scintillation vial. Competitive binding studies. The relative affinity of other compounds for the TCDD receptor in rat hepatic cytosol was measured by their ability to compete with [3H]TCDD for specific binding sites. The general procedure was the same as described above. A concentration of 0.30 mM [3H]TCDD was used, and various concentrations of unlabeled congeners were added to the incubation in 10 CL1p-dioxane immediately after the addition of [3H]TCDD. p-Dioxane (10 ~1) was also added to those tubes which contained only 13H]TCDD or [ 3H JTCDD + TCDF. At this concentration of j3H]TCDD, the specific binding was 15-l 8 times that of nonspecific binding. liquid sci~ti~~atio~ counting and data analysis. Scintillation cocktail (10 ml) was added to each scintillation vial containing 0.2 ml of the incubation mixture or HAP + absolute ethanol, and the radioactivity was determined using a Packard Tri-Carb Model 2450 scintillation spectrometer. Quenching was corrected by automatic external standardization. The tritium counting efficiency was 34-38%. Specific binding was determined as the difference between total binding (tubes containing [3H]TCDD only) and nonspecific binding (tubes containing [ ‘H]TCDD + TCDF). When various concentrations of [3H]TCDD were used, subsequent data was analyzed by the method of Scatchard (36) using linear regression analysis. For the competitive binding studies, the total cytosolic binding of [3H]TCDD in the presence of competitor was calculated and corrected for
4
GASIEWICZ
0
I
2 NUMBER
3 OF
4
5
WASHES
FIG. 1. Binding of [‘HITCDD to hydroxylapatite (HAP). HEDG buffer (0.20 ml) containing approximately 24,000 dpm [ ‘H]TCDD (0) or [‘H JTCDD plus a 200-fold excess of TCDF (0) was incubated with 0.25 ml of a HAP suspension for 30 min at 0-4°C. The HAP, isolated by centrifugation, was washed sequentially with 1.O ml of HEDG buffer or HEDG buffer containing 1% Tween 80. Following centrifugation the HAP was resuspended in 1.0 ml absolute ethanol and the radioactivity associated with the HAP was determined. Each point represents the mean + SD of three determina!ions.
nonspecific binding. Each experiment was performed at least three times using separate animals or pooled samples in the case of mice. In some cases representative results of these experiments are depicted. RESULTS
HAP-[3H]TCDD adsorption. One of the essential requirements of this assay is that unbound [3H]TCDD can be removed from the HAP to the maximum extent possible. Following the incubation of [ 3H]TCDD and [3H]TCDD plus a 200-fold excess of TCDF with the HAP suspension, >99% of the added radioactivity was associated with the
AND NEAL
pellet following centrifugation. Only 2326% of the radioactivity was removed following 5 washes of the HAP with HEDG buffer (Fig. 1). However, following 4 or 5 washes with HEDG buffer containing 1% Tween 80, only 1.7% of the original radioactivity remained associated with the HAP. In addition, there was no significant difference between the amount of radioactivity associated with the HAP incubated with [3H]TCDD or [3H]TCDD + TCDF. Thus, no specific binding of the [3H]TCDD to the HAP could be detected. Essentially, the same results were observed when Triton X100 was substituted for Tween 80 or when the original incubation contained 2 mg bovine serum albumin/ml in addition to the HAP. HAP-[3H]TCDD-receptor adsorption and nonspecific binding. In the development of this assay it was necessary to determine if the [ 3H]TCDD-receptor complex would be adsorbed to the HAP and whether washing would reduce the free and nonspecifically bound [‘H]TCDD without altering the specific binding. Cytosol previously incubated with [3H]TCDD (total binding) or [3H]TCDD + TCDF (nonspecific binding) was added to tubes containing the HAP suspension and incubated for 30 min. Following the incubations, the HAP was washed repeatedly with HEDG buffer containing 1.0% Tween 80. Figure 2 shows that washing with HEDG buffer containing 1% Tween 80 markedly reduced the total and nonspecific binding of [‘H]TCDD but had little effect on specific binding. In this experiment the nonspecific binding was approximately 14% of the specific binding. An additional experiment showed that varying the Tween 80 concentration between 0.05 and 1.0% had little effect upon the amount of specific binding. However, the amount of nonspecific binding observed at 0.05 and 0.1% Tween 80 was approximately twice that observed at 0.5 and 1% (data not shown). As indicated above, after the incubation of the [ 3H]TCDD-labeled hepatic cytosol
ASSAY FOR 2,3,7,8-TETRACHLORODIBENZO-p-DIOXIN
5
RECEPTORS
shows the specific binding of [3H]TCDD in rat hepatic cytosol as a function of protein concentration. Under these conditions the assay of specific binding was linear up to a protein concentration of approximately 3 mg/ml. The departure from linearity beyond this concentration may have resulted from a saturation of the HAP with protein. The linearity of the assay was found to be extended to a protein concentration of 5 mg/ ml if an additional 0.1 ml of the HAP suspension is used in the assay. The lower limit of sensitivity of this assay is, of course, restricted by the specific activity of the I i d [3H]TCDD. In addition, Garola and Mcj i NUMBER OF WASHES Guire (30) have suggested that when very FIG. 2. The effect of washing of HAP on the total, dilute protein concentrations are used in the nonspecific, and specific binding of [‘HITCDD to rat study of estrogen receptors, values for the hepatic cytosol. Rat hepatic cytosol (2.0 ml, 1.9 mg actual number of specific binding sites obprotein/ml) was incubated with [‘H]TCDD (approx 1 X 10’ dpm/ml) or [‘HITCDD plus a 200-fold excess tained by a HAP assay may underestimate of TCDF for 60 min at 20°C. A 0.2-ml aliquot was actual receptor concentrations. taken and incubated with a HAP suspension in HEDG Alteration of specific binding as a funcbuffer for 30 min at 0-4’C. The HAP isolated from the tion of time and temperature of incubation. incubations was sequentially washed with HEDG buffer The specific binding of [3H]TCDD by rat containing 1% Tween 80. Following the final wash the hepatic cytosol reaches a maximum after a HAP was resuspended in 1.0 ml ethanol and the radio1 -h incubation at 20°C and remains constant activity associated with the HAP was determined. Total, specific, and nonspecific binding were determined as for at least 18 h when incubated in the presdescribed under Materials and Methods. Each point ence of [3H]TCDD (Fig. 4). When the inrepresents the mean f SD of three determinations using cubation is carried out at 0°C however, the the same cytosol preparation. The numbers in the hatched area indicate the dpm attributed to specific binding for a particular number of washes.
with HAP followed by centrifugation, over 99% of the radioactivity was found associated with the HAP pellet. In addition, less than 1% of the protein added to the incubation could be detected in the supernatant. The radioactivity that did remain in the supernatant could be completely removed from solution by the addition of 0.2 ml of a dextran-charcoal suspension in HEDG buffer (charcoal, 3%; dextran, 0.03%, w/v). These results suggest that under the conditions of this assay the protein, as well as specifically and nonspecifically bound [3H]TCDD, is nearly completely adsorbed to the HAP. Eflect of protein concentration. Figure 3
42 i
I-/
v)
0
J’ IO
2.0
3.0
PROTEIN CONCENTRATION
4.0 (mg/ml)
50
FIG. 3. Specific binding of [ ‘H]TCDD to rat hepatic cytosol as a function of protein concentration. Rat hepatic cytosol containing various protein concentrations was incubated with [‘H]TCDD or [‘H]TCDD plus a 200-fold excess of TCDF (approx 1 X lo5 dpm/ml) for 60 min at 20°C. Specific binding was determined by the HAP assay as described under Materials and Methods. Each point represents the mean + SD of three determinations using the same cytosol preparation.
GASIEWICZ
AND NEAL
28 24 20 16 12 0 4
TIME (HR)
FIG. 4. The effect of time of incubation and temperature in the presence or absence of ligand on the specific binding of 13H]TCDD to rat hepatic cytosol. Rat hepatic cytosol was incubated at 20 or 0°C in the presence (1.7 mg protein/ml) (0) or absence (1.6 mg protein/ml) (0) of [‘HITCDD and [gH]TCDD plus a 200&d excess of TCDF. At the times indicated, specific binding was determined by the HAP assay as described under Materials and Methods. Each point represents the mean of two determinations using the same cytosol preparation.
same level of specific binding is not reached until 12 to 24 h. Similar results were obtained using cytosol prepared from rat lung and thymus, as well as C57BL/6J mouse liver. Figure 4 also shows the stability of the rat hepatic cytosol receptor when incubated at 0 or 20°C for various periods in the absence of [ 3H JTCDD, followed by the recep-
13Hl TCDD
(dpm x iO~3/0.2mf)
tor assay as described under Materials and Methods. When incubated at 20°C in the absence of TCDD, the receptor rapidly loses its capacity to bind [3H]TCDD. The capacity to specifically bind [3H]TCDD is also decreased by the incubation of the cytosol at 0°C in the absence of TCDD, although at a slower rate. Additional studies (data not
BOUND
TCDD
ffmol/mg
protein)
FIG. 5. Receptor saturation studies. The specific binding of [‘HITCDD to hepatic cytosol (1.73 mg protein/ml) was determined by the HAP assay as described under Materials and Methods. (A) Representative data showing the total, nonspecific, and specific binding of 13H]TCDD to rat hepatic cytosol at various concentrations of [3H]TCDD, (B) Scatchard plot of the specific binding data in (A). KD = 0.16 nhr; n = 69 fmol/mg protein. Each point is the mean of two determinations using the same cytosol preparation.
ASSAY FOR 2,3,7,8-TETRACHLORODIBENZO-p-DIOXIN TABLE
1
CONCENTRATION (n) AND DESOCIA~ON CONSTANTS (KD)OF THE TCDD RECWTOR IN RAT AND MOUSE HEPATIC CYTOSOL
(fmo?,mg Species
Sprague-Dawley rat (7) CS7BL/6J mice May-July (4) February-April (3) DBA/2J mice (3) B6D2 F,/J mice February-April (3)
protein) 61+
(2, 5
0.12 f 0.03
74 t 10 47~~ 8
0.29-t 0.01 0.29k 0.03
235 2
0.42 + 0.03
-=
-0
Note. Each value represents the mean + SD of (x) determinations. Separate determinations using the same cytosol were performed in duplicate. a Unable to be determined. See text and Fig. 6.
shown) demonstrated that these results were due to a decrease in the number of receptor sites rather than to an effect upon the apparent affinity (Kn). Similar results have been observed for specific binding sites in mouse hepatic cytosol incubated with TCDD ( 14) or for steroid hormone receptors incubated in the presence of the steroid (37,38). Demonstration of saturability. Figure 5A shows the saturation curve resulting from the incubation of rat hepatic cytosol with various concentrations of [3H]TCDD and [ 3H]TCDD plus a 200-fold excess of TCDF. In this experiment, the specific binding was 7- 13 times that of the nonspecific binding. Figure 5B is a Scatchard plot (36) of the same data. For this preparation the equilibrium dissociation constant KD was 0.16 nM, and the total number of specific binding sites for TCDD was 69 fmol/mg cytosolic protein. Similar results were obtained using MDEG buffer (25 mM MOPS, pH 7.4; 1 mM dithiothreitol; 1 mM EDTA; 5% glycerol) or KTMD buffer (100 mM KCI; 25 mM Tris-HCl, pH 7.4; 5 mM MgCl,; 1 mM dithiothreitol). Table 1 shows the results of analysis of the hepatic cytosolic receptor in Sprague-
RECEPTORS
7
Dawley rats, as well as C57BL/6J, DBA/ 23, and B6D2FI/J mice. In these experiments E3H]TCDD concentrations of 0.1 to 1.0 nM were used, and with the exception of hepatic cytosol from DBA/2J mice, the specific binding was greater than 4 times that of the nonspecific binding. The cytosol from rat liver contained binding sites with the highest affinity, 0.12 nM. Some apparent seasonal variation in the number of specific binding sites was observed in C57BL/6J mice. Seasonal variations in mouse hepatic cytosol receptor number also were reported by Okey et al. (28). Little specific binding was observed using the hepatic cytosol from DBA/U (Table 1, Fig. 6). With this strain of mice, the specific binding was 20-40s of the nonspecific binding and was considered too low to accurately quantitate the number of receptors or affinity. The hepatic cytosol from the B6D2FI/J cross showed somewhat intermediate specific binding as compared to the C57BL/6J or the DBA/2J parent strains (Table 1, Fig. 6).
13H] TCDD
(dpm x 10 3/0.2ml)
FIG. 6. Specific binding of [ ‘HJTCDD to the hepatic cytosol from C57BL/6J, DBA/2J, and B6D2F1/J mice. Specitic binding was determined by the HAP assay as described under Materials and Methods. The protein concentration for C57BL/6J mice (obtained FebruaryApril) was 1.70 mg/ml, for DBA/ZJ mice was 2.25 mg/ ml, and for B6D2F,/J mice (obtained February-April) was 2.10 mg/ml. Each point represents the mean of two determinations using the same cytosol preparation.
8
GASIEWICZ
AND NEAL
8 100 3%
90 80
5
70
8
60
5
50
g 2
do 30
iii g
20 10
s
lo-"
1O-9 CONCENTF3ATlON
Ida
id7 OF COMPETITOR
(M)
FIG. 7. Competition by dibenzo-p-dioxin congeners and other compounds for the specific binding of [3H]TCDD to rat hepatic cytosoi. The specific binding of 13H]TCDD to rat hepatic cytoso1 (2.15 mg protein/mi) was determined by the HAP assay as described under Materials and Methods. The concentration of [“HITCDD was 0.30 nM. The concentrations of other compounds is given on the abscissa.
Competitive binding by congeners of TCDD and other compounds. Figure 7 demonstrates that the specific binding of 13H]TCDD to rat hepatic cytosol as determined by the HAP assay is competitively inhibited by other compounds known to induce a pattern of monooxygenase activities which is similar to those induced by TCDD. The results are similar to those obtained previously using hepatic cytosol from C57BLf6J mice (14). As expected, the apparent binding affinities of 1,2,4-trichlorodibenzo-p-dioxin and dibenzo-p-dioxin for the rat hepatic cytosol receptor are 2.5 to 4 orders of magnitude lower than TCDD or TCDF. Phenobarbital, which induces a different pattern of microsomal monooxygenases from those induced by TCDD (6), shows no competitive binding at concentrations up to 1 X lo+ M. DISCUSSION
The results of these studies have shown that the HAP assay gives reliable and reproducible estimates of the number of specific TCDD binding sites in mouse and rat hepatic cytosol and their relative affinities for TCDD and related compounds. A higher ratio of specific to nonspecific binding is the
major advantage of this assay system over the previously published dextran-charcoal method (14), although the results obtained by the two methods are quite similar. The present method is also faster and appears to give more consistent data than the use of sucrose gradient techniques (28,29). The present method appears to have similar advantages over the isoelectric focusing procedure (27), which also requires trypsin treatment of the cytosol to prevent receptor aggregation. Utilizing the isoelectric focusing technique, Carlstedt-Duke and coworkers (27,39) reported concentrations of the receptor in hepatic cytosol from Sprague-Dawley rats of 14 to 31 fmol/mg protein with a Kn of 0.61 nM. These investigators incubated the cytosol with [3H]TCDD or [3H]TCDD + TCDF for 1 to 2 h at 0°C. Using the HAP assay under the same conditions, receptor concentrations of 20 to 30 fmol/mg protein were observed with Kn’s ranging from 0.2 to 0.8 nrvr. Additional experiments indicated that, at O”C, equilibration of the receptor in rat hepatic cytosol with added [‘H]TCDD or TCDF does not occur until 12 to 24 h (Fig. 4). Similar results were obtained using cytosol prepared from rat lung and thymus,
ASSAY FOR 2,3,7,8-TETRACWLORODIBENZO-P-DIOXIN
as well as hepatic cytosol from C57BL/6J mice. Thus, it is possible that, due to incomplete equilibration of the receptor with [ 3H]TCDD, the investigations by CarlstedtDuke and co-workers (27,39) may have underestimated the number of receptors and their relative affinity for TCDD. However, it should be pointed out that the times to equilibration observed in our laboratory differ considerably from those previously obtained by other investigators. Using a dextran-charcoal assay, Poland et al. (14) reported that constant specific binding is reached by 1 h at 0°C. Utilizing sucrose gradient techniques, Okey et al. (28) reported nearly complete equilibration within 5 min at 4°C. The reasons for these differences are as yet unknown. Hepatic cytosol from both rats and mice appear to reach constant specific binding rapidly at 20°C. In the present study, similar results for the number of specific binding sites and Kn values were obtained when the same rat hepatic cytosol preparation was incubated with various concentrations of E3H]TCDD and [3H]TCDD + TCDF for 2 h at 20°C or 24 h at 0°C. Hannah et al. (29) reported the Ah receptor in C57BLf6J mice to have an approximate molecular weight of 245,000. Using high-resolution gel filtration, we have determined the molecular weight of the specific binding peak in rat hepatic cytosol to be approximately 280,000.4 The specific binding observed by this gel-filtration method corresponds to the specific binding as determined by the assay presently described utilizing HAP. These preliminary studies suggest, but no not prove, that the specific binding as determined by the HAP assay is qualitatively comparable to that determined by other assay systems. Utilizing a sucrose gradient technique, Okey et al. (28) reported a Kn of 0.7 nM for TCDD specific binding sites in hepatic cytosol from C57BL/6J mice. Examination of 4 Gasiewicz, T. A., and Rucci, G., in preparation.
RECEPTORS
9
t3H]TCDD from the same supplier using high-~rformance liquid chromatography (33) revealed the [ 3H]TCDD was only about 65% pure. The main contaminating radioactive peaks had the same retention time as the tri- and pentachlorodibenzo-p-dioxin isomers. Thus, it is possible that the higher Kn value obtained by Okey et al. (28) may be in part a result of the presence of radiolabeled dioxin contaminants having a lower afiinity for the receptor. The previous investigations by Poland et al. ( 14) and Okey et ai. (28) reported either very low or unmeasurable specific binding of [ 3H]TCDD to hepatic cytosol from DBA/ 25 (nonresponsive) mice. Similar results were obtained in the present study. At present, adequate analysis of the specific binding in the DBA/U mouse hepatic cytosol cannot be performed due to the limited solubility as well as to the specific activity of the available E3H]TCDD. It is of interest that in hepatic cytosol preparations from B6D2 F,/J mice (offspring of C57BL/6J and DBA/2J parents, heterozygous at the Ah locus), the total amount of specific binding (Table 1, Fig. 6) is somewhat intermediate between that of the C57BL/6J and DBA/2J mice. However, the relative affinity for TCDD is only slightly different from that observed in C57BL/6J hepatic cytosol (Table 1). Intermediate responses of the B6D2FI/J offspring to TCDD have been observed for AHH induction (40), cleft palate production (15), thymic atrophy (151, and lethality.’ Although indirect evidence ( 14,16) does suggest that the major defect in the DBA/U mice is an altered receptor with reduced affinity toward TCDD, from the present data it is not possible to determine whether the low amount of specific binding observed in DBA/2.J mouse is due to a decrease in the number of receptor molecules, a decreased affinity, or a combination of these factors. It is also possible that the induction of AHH ’ Gasiewicz, T. A., Geiger, L. E., and Neal, R. A., in preparation.
10
GASIEWICZ
activity in nonresponsive mice by TCDD is initiated by a mechanism different than binding to this receptor protein in the cytoso1. Com~tition studies (Fig. 7) suggest that the specificity of binding of various compounds to the receptor species in SpragueDawley rats is quantitatively similar to that observed in the hepatic cytosol from C57BL/ 65 mice (14). These data support but are not suf?icient evidence for the hypothesis that, as in mice ( 14), the specific cytosolic binding of TCDD to the receptor in rats mediates AHH induction. Additional support comes from the observation that the ability of congeners of TCDD and other com~unds such as 3-methylcholanthrene and benzo[cu]pyrene to induce AHH activity is similar in both rats and mice (40-43). ACKNOWLEDGMENTS The excellent technical assistance of George Rucci is greatly appreciated. This study was supported in part by Grants ES-02515, ES-00267, ES-015.52, and ES05100 and Center Grant ES-01247 from the National Institute of Environmental Health Sciences, BRSG Grant RR 05403, and a grant from the Pharmaceutical Manufactures Association Foundation. This paper is also based on work partially performed under Contract DE-AC02-76EV03490 with the U. S. Department of Energy at the University of Rochester Department of Radiation Biology and Biophysics and has been assigned Report No. UR-3490-2035.
REFERENCES 1. Schwetz, B. S., Norris, J. M., Sparschu, G. L., Rowe, V. K., Gehring, P. J., Emerson, J. L., and Gerbig, G. G. (1973) Environ. Health Perspec. 5, 87-99. 2. Goldstein, J. A. (1979) Ann. N. Y. Acud. Sci. 320, 164-178. 3. Kimbrough, R. D. (1974) CRC Crir. Rev. Toxicoi. 2,445-498.
McConnell, E. E., and Moore, J. A. (1979) Ann. N. I’. Acad. Sci. 320, 138-150. 5. Poland, A., Greenlee, W. F., and Kende, A. S. (1979) Ann. N. Y. Acad. Sci. 320, 214-230. 6. Nebert, D. W., and Jensen, N. M. (1979) CRC Act. Rev. Biochem. 6,401-437. 7. Negishi, M., and Nebert, D. W. (1979) J. Biol. Chem. 254, 11,015-l 1,023. 4.
AND NEAL 8. Owens, I. S. (1977)J. Biol. Chem. 252,2827-2833. 9. Kumaki, K., Jensen, H. M., Shire, J. 0. M., and Nebert, D. W. (1977) J. Biol. Chem. 252, 157165. IO. Nebert, D. W., Jensen, N. M., Perry, J. W., and Oka, T. (1980) J. Biol. Chem. 255,6836-6842. 11. Gielen, J.E., Goujon, F. M., and Nebert, D. W. (1972) J. Biol. Chem. 247, 1125-l 137. 12. Nebert, D. W., and Gielen, J. E. (1972) Fed. Proc. 31, 1315-1325. 13. Thomas, P. E., Kouri, R. E., and Hutton, J. J. (1972) Biochem. Genet. 6, 157-168. 14. Poland, A., Glover, E., and Kende, A. S. f 1976) J. Biol. Chem. 251,4936-4946. 15. Poland, A., and Clover, E. (1980) Mol. Pharmacol. 17, 86-94.
16. Greenlee, W. F., and Poland, A. (1979) J. Biof. Chem. 254,9814-9821. 17. Kouri, R. E., and Nebert, D. W. (1977) in Origins of Human Cancer (Watson, J. D., and Hiatt, H., eds.), pp. 81 l-835, Cold Spring Laboratory, New York. 18. Kouri, R. E., Ratrie, H., and Whitmire, C. E. (1973) J. Nat. Cancer inst. 51, 197-200. 19. Kociba, R. J., Keyes, D. G., Beyer, J. E., Carreon, R. H., Wade, C. E., Dittenber, D. A., Kalnins, R. P., Frauson, L. E., Park, C. N., Barnard, S. D., Hummel, R. A., and Humiston, C. G. (1978) Toxicol. Appl. Pharmacol. 46,279-303. 20. Toth, R., Sugar, J., Somfai-Felle, S., and Bence, J. (1978) Progr. Biochem. Pharmacof. 14, 8293. 21. VanMiller, J. P., Lalich, J. J., and Allen, J. R. (1977) Chemosphere 6,537-544. 22. Poland, A., and Glover, E. (1979) Cancer Res. 39, 3341-3344. 23. Geiger, L. E., and Neal, R. A. (1981) Toxicol. Appl. Pharmacol. 59, 125-l 29. 24. Nebert, D. W., Thorgeirsson, S. S., and Felton, J. S. (1976) in In Vitro Metabolic Activation in Mutagenesis Testing (DeSerres, F. J., Fouts, J. R., Bend, J. R., and Philpot, R. M., eds.), pp. 105-124, Elsevier-North-Holland, Amsterdam/ New York. 25. Wassom, J. S., Huff, J. E., and Laprieno, N. ( 1978) Mutat. Res. 47, 141-160. 26. Pitot, H. C., Goldsworthy, T., Campbell, H. A., and Poland, A. (1980) Cancer Res. 40, 3616-3620. 27. Carlstedt-Duke, J., Elfstrom, G., Snochowski, M., Hogberg, B., and Gustafsson, J. (1978) Toxicol. Lett. 2, 365-373. 28. Okey, A. B., Bondy, G. P., Mason, T. M., and Nebert, D. W. (1979) J. Biol. Chem. 254, 11,636-l 29.
1,648.
Hannah, R. R., Nebert, D. W., and Eisen, H. J. (1981) J. Biol. Chem. 256,4584-4590.
ASSAY FOR 2,3,7,8-TETRA~HLORODIB~NZO-~DIOXIN 30. Garola, R. E., and McGuire, W. L. (1978) Cancer Res. 38,2216-2220. 31. Pavlii, E. J., and Coulson, P. B. (1976) J. Steroid Biochem. 7, 357-368. 32. Olson, J. R., Gasiewicz, T. A., and Neal, R. A. (1980) Toxicol. Appl. Pharmacol. 56, 78-85. 33. Gasiewicz, T. A., and Neal, R. A. (1979) Toxicof. Appl. Pharmacol. 51, 329-339. 34. Waddell, W. J. (1956)J. Lab. Cfin. Med. 48, 311314. 35. Lowry, 0. H., Rosebrough, H. J., Farr, A. L., and Randall, R. J. (1951) J. &of. Chem. 193, 265275. 36. Scatchard, G. (1949) Ann. N. Y. ACQ~. Sci. 51, 660-672.
RECEPTORS
11
37. Gorski, J., and Gannon, F. (1976) Annu. Rev. Physiol. 38,425-450. 38. Rousseau, G. G., Higgins, S. J., Baxter, J. D., Gelfand, D., and Tomkins, G. M. (1975) .I. Biol. Chem. 250,6015-6021. 39. Carlstedt-Duke, J. M. B. (1979) Cancer Res. 39, 3172-3176. 40. Poland, A., and Glover, E. (1975) Mol. Pharmacol. 11, 389-398. 4 1. Poland, A. P., and Glover, E. (1973) Mol. Pharmacol. 9, 736-747. 42. Poland, A. P., and Glover, E. (1974) Mol. Pharmacol. 10, 349-359. 43. Poland, A. P., Glover, E., Robinson, J. R., and Nebert, D. W. (1974) J. Biol. Chem. 249,55995606.
BIOCHEMISTRY
124,
I- 11 ( 1982)
The Examination and Quantitation of Tissue Cytosolic Receptors 2,3,7,8-Tetrachlorodibenzo-pdioxin using Hydroxylapatite T. A. GASIEWICZ’
for
AND R. A. NEAL*-’
Department of Radiation Biology and Biophysics, The University of Rochester, Rochester, New York 14642, and *Department of Biochemistry, Vanderbilt University, Nashville. Tennessee 37232 Received November 20, 1981 An assay using hydroxylapatite has been developed for the examination and quantitation of cytosolic receptors for 2,3,7,8-tetrachlor~ibenzo”~-dioxin (TCDD). This method, as compared to others, has a relatively high ratio of specific to nonspecific binding of [3H]TCDD and is relatively rapid. The total number of specific binding sites and equilibrium dissociation constants (Kn) for [‘H]TCDD were determined in hepatic cytosol from Sprague-Dawley rats and C57BL/6J, DBA/ZJ, and B6D2FI/J mice. With the exception of the cytosol from DBA/ 25 mice in which little specific binding was observed, high-affinity, specific binding was obtained in ail cases. The rat hepatic cytosol demonstrated the highest affinity for [‘H]TCDD with a Kn of 0.12 nM. The total amount of specific [3H]TCDD binding in hepatic cytosol from B6D2F,/J mice was found to be intermediate between that of the C57BL/6J and DBA/ZJ parents. Ligand competition studies suggest the specificity of binding of various compounds to the receptor species from Sprague-Dawley rats is similar to that observed with the hepatic cytosol from C57BL/6J mice. These data support the hypothesis that, as in mice, the particular cytosol binding species in rats is the receptor that may mediate the induction of aryl hydrocarbon hydroxylase activity.
TCDD3 is formed as a contaminant in the synthesis of 2,4,5-trichlorophenol and is considered on both an acute and chronic basis to be one of the most toxic synthetic compounds known (1). This compound is a representative of a group of structurally related halogenated aromatic hydr~arbons ineluding other dibenzo-p-dioxins, biphenyls, dibenzofurans, as well as the azo- and azoxybenzenes, all of which produce similar patterns of toxicity and bi~hemi~al responses (2-5). The mechanism(s) of action of these compounds is unknown.
TCDD has been shown to be a potent inducer of two or more forms of cytochrome P-450 and approximately 20 associated monooxygenase activities (including aryl hydrocarbon hydroxylase (AHH) (6,7), as well as microsomal UDP-glucuronosyltransferase (S), cytosolie reducedNAD(P):menadione oxidoreductase (91, and ornithine decarboxylase (10). In certain strains of mice, the induction of these enzymes is regulated by a single genetic locus, the Ah locus ( 11-l 3). The stereo-specific, high-affinity binding of TCDD and its congeners to a cytosolic receptor protein appears to correlate with the ability of these compounds to induce AHH activity (responsiveness) (14), as well as elicit toxic effects in various animal species (2,15). The translocation of this TCDD-receptor complex to the nucleus, subsequent nuclear binding, and alteration of gene expression appear to be essential events in the TCDD-mediated in-
’ To whom requests for reprints should be addressed. * Present address: Chemical Industry Instituteof Toxicology, P.O. Box 12137, Research Triangle Park, N. C. 27709. 3 Abbreviations used: TCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxin; AHH, aryl hydr~arbon hydroxylase; HAP, hydeoxylapatite; TCDF, 2,3,7,8-tetrachlorodibenzofuran; Hepes, 4-( 2-hydroxyethyl)- 1-piperazineethanesulfonic acid, MOPS, morpholinopropanesulfonic acid. 1
0003-2697/82/l
10001-l 1$02.00/O
Copyright 0 1982 by Academic Press, Inc. All rights of reproduction in any form reserved.
2
GASIEWICZ
duction of AHH activity (16). Aromatic hydrocarbon hydroxylase activity regulated by the Ah locus appears to be a significant step in the metabolism of certain polycyclic aromatic hydrocarbons (6) and may alter their ability to induce carcinogenesis in various strains of mice (17,18). TCDD has been reported to be a potent carcinogen in chronic feeding studies using rats and mice (19-21). However, the covalent binding of TCDD and/or its metabolites to tissue macromolecules was found to be 4 to 6 orders of magnitude lower than most chemical carcinogens (22). Recent studies suggest TCDD is not mutagenic in bacterial systems (23-25). Studies by Pitot et al. (26) indicate that TCDD is an extremely potent promoting agent. It has been proposed that its action as a promoter may be mediated by its binding to a receptor and the subsequent alteration in gene expression (26). Previous investigators have utilized dextran-charcoal adsorption techniques (14), isoelectric focusing (27) and sucrose-density gradients (28,29) for the assay and examination of TCDD cytosolic receptors. These methods possess a number of disadvantages including a high level of nonspecific binding of [ 3H]TCDD, inconsistency, possible alteration of the receptor by trypsinization, as well as being time consuming. In this report, a method is described for the quantitation of the cytosolic receptor for TCDD utilizing hydroxylapatite (HAP). Similar procedures using HAP have been applied to the study of steroid receptors (30,31). We have found this method to be specific and relatively rapid for the study of TCDD receptors. Additional properties of these receptors in rats and mice are also described. MATERIALS
AND METHODS
Materials. [ 1,6-3H]TCDD (50.5 mmol) was synthesized and purified as viously described (32). The compound >98% chemically pure as determined
Ci/ prewas by
AND NEAL
high-performance liquid chromatography (33). TCDF was a generous gift of Dr. J. A. Moore (NIEHS, Research Triangle Park, N. C.). Benzo[cu Jpyrene, 3-methylcholanthrene, Triton X- 100, polyethylene sorbitan monooleate (Tween 80), dithiothreitol, Hepes, and MOPS were purchased from Sigma Chemical Company (St. Louis, MO.). BNaphthoflavone was purchased from Aldrich Chemical Company (Milwaukee, Wis.). 1,2,4-Trichlorodibenzo-p-dioxin and dibenzo-p-dioxin were obtained from Analabs (North Haven, Conn.). 1,2,3,4,7,8Hexachlorodibenzo-p-dioxin was purchased from KOR Isotopes (Cambridge, Mass.). DNA-grade HAP was purchased from BioRad Laboratories (Rockville Centre, N. Y.). Decolorizing charcoal (Neutral Norit A) was obtained from Fisher Scientific (Rochester, N. Y.). Liquescint scintillation cocktail was purchased from National Diagnostics (Somerville, N. Y.). Animals. Male Sprague-Dawley rats (200-250 g) were purchased from Charles River (Wilmington, Mass.). Male C57BL/ 65, DBA/2J, and B6D2F1/J mice (20-22 g) were obtained from Jackson Laboratories (Bar Harbor, Me.). Animals were housed under laboratory conditions for at least 3 days prior to use. They were fed commercial rat or mouse chow (Ralston Purina, Richmond, Ind.) and water ad libitum. Tissue preparation. Animals were killed by decapitation or cervical dislocation and their livers were excised and weighed. These tissues were homogenized in 3 vol of HEDG buffer (25 mM Hepes, pH 7.4; 1.5 mM EDTA; 1 mM dithiothreitol; and 10% glycerol (v/v)) in a glass homogenizer fitted with a Teflon pestle. For mice, 2 or 3 livers were pooled for a single preparation. Unless specified, these and the remaining procedures were carried out at 0-4°C. Each homogenate was transferred to polycarbonate tubes and centrifuged at 10,OOOg for 20 min. The supernatant fraction was collected and centrifuged at 105,OOOg for 60 min. Following each centrifugation the turbid lipid layer was
ASSAY
FOR
2,3,7,8-TETRACHLORODIBENZO-p-DIOXIN
removed with a Pasteur pipet. Following the last centrifugation the cytosol was removed, taking special care not to disturb the microsomal pellet. The protein concentrations of the hepatic cytosol preparations were estimated by the absorbance at 215/225 nm (34) and later confirmed by the method of Lowry er al. (35) using bovine serum albumin as a standard. The protein concentration was diluted with HEDG buffer to between 1.5 and 2.5 mg/ml. The diluted cytosol was used within 1 h after preparation. HAP standard aiming assay. DNA-grade HAP was washed with HEDG buffer, pH 7.4, until the pH of the washes remained at 7.4. The HAP was then resuspended in 2 vol of HEDG buffer at 0-4°C with gentle shaking. The incubation conditions for this assay are similar to that previously described by Poland et al. (14). Two milliliters of the 105,OOOg supernatant fraction with a protein concentration of 1.5 to 2.5 mg/ml was incubated with various concentrations of [3H]TCDD (0.1-1.0 nM) and with [3H]TCDD plus a 200-fold excess of unlabeled TCDF for 2 h at 20°C in a shaker water bath. The [ 3H JTCDD and [ 3H]TCDD + TCDF were added to the incubations in a volume of 5 ~1 p-dioxanelml of cytosol. After 2 h, 0.2 ml of the incubation was removed and added to a scintillation vial, and the total [3H]TCDD contained per volume of incubation mixture was determined. An additional 0.2 ml of this mixture was also removed and added to a disposable 13 X 1OOmm glass test tube containing 0.25 ml of the HAP suspension. These were incubated for 30 min at 0-4°C with gentle shaking every 10 min. At the end of this time, 1.0 ml of ice-cold HEDG buffer containing 0.5% (v/ v) of Tween 80 was added, These tubes were shaken in a Vortex mixer and centrifuged at 1500 rpm for 5 min. The HAP pellet to which the cytosolic protein had been adsorbed was washed three additional times with 1.0 ml of the HEDG buffer containing
RECEPTORS
3
Tween 80. Following each centrifugation the supernatant was drawn off using a Pasteur pipet. After the last wash, 1.O ml of absolute ethanol was added to the tube containing the HAP pellet. The contents were mixed using a Vortex mixer, removed with a Pasteur pipet, and added to scintillation vials. The tubes and the pipets were washed with an additional 1.0 ml of absolute ethanol, and this wash was also added to the scintillation vial. Competitive binding studies. The relative affinity of other compounds for the TCDD receptor in rat hepatic cytosol was measured by their ability to compete with [3H]TCDD for specific binding sites. The general procedure was the same as described above. A concentration of 0.30 mM [3H]TCDD was used, and various concentrations of unlabeled congeners were added to the incubation in 10 CL1p-dioxane immediately after the addition of [3H]TCDD. p-Dioxane (10 ~1) was also added to those tubes which contained only 13H]TCDD or [ 3H JTCDD + TCDF. At this concentration of j3H]TCDD, the specific binding was 15-l 8 times that of nonspecific binding. liquid sci~ti~~atio~ counting and data analysis. Scintillation cocktail (10 ml) was added to each scintillation vial containing 0.2 ml of the incubation mixture or HAP + absolute ethanol, and the radioactivity was determined using a Packard Tri-Carb Model 2450 scintillation spectrometer. Quenching was corrected by automatic external standardization. The tritium counting efficiency was 34-38%. Specific binding was determined as the difference between total binding (tubes containing [3H]TCDD only) and nonspecific binding (tubes containing [ ‘H]TCDD + TCDF). When various concentrations of [3H]TCDD were used, subsequent data was analyzed by the method of Scatchard (36) using linear regression analysis. For the competitive binding studies, the total cytosolic binding of [3H]TCDD in the presence of competitor was calculated and corrected for
4
GASIEWICZ
0
I
2 NUMBER
3 OF
4
5
WASHES
FIG. 1. Binding of [‘HITCDD to hydroxylapatite (HAP). HEDG buffer (0.20 ml) containing approximately 24,000 dpm [ ‘H]TCDD (0) or [‘H JTCDD plus a 200-fold excess of TCDF (0) was incubated with 0.25 ml of a HAP suspension for 30 min at 0-4°C. The HAP, isolated by centrifugation, was washed sequentially with 1.O ml of HEDG buffer or HEDG buffer containing 1% Tween 80. Following centrifugation the HAP was resuspended in 1.0 ml absolute ethanol and the radioactivity associated with the HAP was determined. Each point represents the mean + SD of three determina!ions.
nonspecific binding. Each experiment was performed at least three times using separate animals or pooled samples in the case of mice. In some cases representative results of these experiments are depicted. RESULTS
HAP-[3H]TCDD adsorption. One of the essential requirements of this assay is that unbound [3H]TCDD can be removed from the HAP to the maximum extent possible. Following the incubation of [ 3H]TCDD and [3H]TCDD plus a 200-fold excess of TCDF with the HAP suspension, >99% of the added radioactivity was associated with the
AND NEAL
pellet following centrifugation. Only 2326% of the radioactivity was removed following 5 washes of the HAP with HEDG buffer (Fig. 1). However, following 4 or 5 washes with HEDG buffer containing 1% Tween 80, only 1.7% of the original radioactivity remained associated with the HAP. In addition, there was no significant difference between the amount of radioactivity associated with the HAP incubated with [3H]TCDD or [3H]TCDD + TCDF. Thus, no specific binding of the [3H]TCDD to the HAP could be detected. Essentially, the same results were observed when Triton X100 was substituted for Tween 80 or when the original incubation contained 2 mg bovine serum albumin/ml in addition to the HAP. HAP-[3H]TCDD-receptor adsorption and nonspecific binding. In the development of this assay it was necessary to determine if the [ 3H]TCDD-receptor complex would be adsorbed to the HAP and whether washing would reduce the free and nonspecifically bound [‘H]TCDD without altering the specific binding. Cytosol previously incubated with [3H]TCDD (total binding) or [3H]TCDD + TCDF (nonspecific binding) was added to tubes containing the HAP suspension and incubated for 30 min. Following the incubations, the HAP was washed repeatedly with HEDG buffer containing 1.0% Tween 80. Figure 2 shows that washing with HEDG buffer containing 1% Tween 80 markedly reduced the total and nonspecific binding of [‘H]TCDD but had little effect on specific binding. In this experiment the nonspecific binding was approximately 14% of the specific binding. An additional experiment showed that varying the Tween 80 concentration between 0.05 and 1.0% had little effect upon the amount of specific binding. However, the amount of nonspecific binding observed at 0.05 and 0.1% Tween 80 was approximately twice that observed at 0.5 and 1% (data not shown). As indicated above, after the incubation of the [ 3H]TCDD-labeled hepatic cytosol
ASSAY FOR 2,3,7,8-TETRACHLORODIBENZO-p-DIOXIN
5
RECEPTORS
shows the specific binding of [3H]TCDD in rat hepatic cytosol as a function of protein concentration. Under these conditions the assay of specific binding was linear up to a protein concentration of approximately 3 mg/ml. The departure from linearity beyond this concentration may have resulted from a saturation of the HAP with protein. The linearity of the assay was found to be extended to a protein concentration of 5 mg/ ml if an additional 0.1 ml of the HAP suspension is used in the assay. The lower limit of sensitivity of this assay is, of course, restricted by the specific activity of the I i d [3H]TCDD. In addition, Garola and Mcj i NUMBER OF WASHES Guire (30) have suggested that when very FIG. 2. The effect of washing of HAP on the total, dilute protein concentrations are used in the nonspecific, and specific binding of [‘HITCDD to rat study of estrogen receptors, values for the hepatic cytosol. Rat hepatic cytosol (2.0 ml, 1.9 mg actual number of specific binding sites obprotein/ml) was incubated with [‘H]TCDD (approx 1 X 10’ dpm/ml) or [‘HITCDD plus a 200-fold excess tained by a HAP assay may underestimate of TCDF for 60 min at 20°C. A 0.2-ml aliquot was actual receptor concentrations. taken and incubated with a HAP suspension in HEDG Alteration of specific binding as a funcbuffer for 30 min at 0-4’C. The HAP isolated from the tion of time and temperature of incubation. incubations was sequentially washed with HEDG buffer The specific binding of [3H]TCDD by rat containing 1% Tween 80. Following the final wash the hepatic cytosol reaches a maximum after a HAP was resuspended in 1.0 ml ethanol and the radio1 -h incubation at 20°C and remains constant activity associated with the HAP was determined. Total, specific, and nonspecific binding were determined as for at least 18 h when incubated in the presdescribed under Materials and Methods. Each point ence of [3H]TCDD (Fig. 4). When the inrepresents the mean f SD of three determinations using cubation is carried out at 0°C however, the the same cytosol preparation. The numbers in the hatched area indicate the dpm attributed to specific binding for a particular number of washes.
with HAP followed by centrifugation, over 99% of the radioactivity was found associated with the HAP pellet. In addition, less than 1% of the protein added to the incubation could be detected in the supernatant. The radioactivity that did remain in the supernatant could be completely removed from solution by the addition of 0.2 ml of a dextran-charcoal suspension in HEDG buffer (charcoal, 3%; dextran, 0.03%, w/v). These results suggest that under the conditions of this assay the protein, as well as specifically and nonspecifically bound [3H]TCDD, is nearly completely adsorbed to the HAP. Eflect of protein concentration. Figure 3
42 i
I-/
v)
0
J’ IO
2.0
3.0
PROTEIN CONCENTRATION
4.0 (mg/ml)
50
FIG. 3. Specific binding of [ ‘H]TCDD to rat hepatic cytosol as a function of protein concentration. Rat hepatic cytosol containing various protein concentrations was incubated with [‘H]TCDD or [‘H]TCDD plus a 200-fold excess of TCDF (approx 1 X lo5 dpm/ml) for 60 min at 20°C. Specific binding was determined by the HAP assay as described under Materials and Methods. Each point represents the mean + SD of three determinations using the same cytosol preparation.
GASIEWICZ
AND NEAL
28 24 20 16 12 0 4
TIME (HR)
FIG. 4. The effect of time of incubation and temperature in the presence or absence of ligand on the specific binding of 13H]TCDD to rat hepatic cytosol. Rat hepatic cytosol was incubated at 20 or 0°C in the presence (1.7 mg protein/ml) (0) or absence (1.6 mg protein/ml) (0) of [‘HITCDD and [gH]TCDD plus a 200&d excess of TCDF. At the times indicated, specific binding was determined by the HAP assay as described under Materials and Methods. Each point represents the mean of two determinations using the same cytosol preparation.
same level of specific binding is not reached until 12 to 24 h. Similar results were obtained using cytosol prepared from rat lung and thymus, as well as C57BL/6J mouse liver. Figure 4 also shows the stability of the rat hepatic cytosol receptor when incubated at 0 or 20°C for various periods in the absence of [ 3H JTCDD, followed by the recep-
13Hl TCDD
(dpm x iO~3/0.2mf)
tor assay as described under Materials and Methods. When incubated at 20°C in the absence of TCDD, the receptor rapidly loses its capacity to bind [3H]TCDD. The capacity to specifically bind [3H]TCDD is also decreased by the incubation of the cytosol at 0°C in the absence of TCDD, although at a slower rate. Additional studies (data not
BOUND
TCDD
ffmol/mg
protein)
FIG. 5. Receptor saturation studies. The specific binding of [‘HITCDD to hepatic cytosol (1.73 mg protein/ml) was determined by the HAP assay as described under Materials and Methods. (A) Representative data showing the total, nonspecific, and specific binding of 13H]TCDD to rat hepatic cytosol at various concentrations of [3H]TCDD, (B) Scatchard plot of the specific binding data in (A). KD = 0.16 nhr; n = 69 fmol/mg protein. Each point is the mean of two determinations using the same cytosol preparation.
ASSAY FOR 2,3,7,8-TETRACHLORODIBENZO-p-DIOXIN TABLE
1
CONCENTRATION (n) AND DESOCIA~ON CONSTANTS (KD)OF THE TCDD RECWTOR IN RAT AND MOUSE HEPATIC CYTOSOL
(fmo?,mg Species
Sprague-Dawley rat (7) CS7BL/6J mice May-July (4) February-April (3) DBA/2J mice (3) B6D2 F,/J mice February-April (3)
protein) 61+
(2, 5
0.12 f 0.03
74 t 10 47~~ 8
0.29-t 0.01 0.29k 0.03
235 2
0.42 + 0.03
-=
-0
Note. Each value represents the mean + SD of (x) determinations. Separate determinations using the same cytosol were performed in duplicate. a Unable to be determined. See text and Fig. 6.
shown) demonstrated that these results were due to a decrease in the number of receptor sites rather than to an effect upon the apparent affinity (Kn). Similar results have been observed for specific binding sites in mouse hepatic cytosol incubated with TCDD ( 14) or for steroid hormone receptors incubated in the presence of the steroid (37,38). Demonstration of saturability. Figure 5A shows the saturation curve resulting from the incubation of rat hepatic cytosol with various concentrations of [3H]TCDD and [ 3H]TCDD plus a 200-fold excess of TCDF. In this experiment, the specific binding was 7- 13 times that of the nonspecific binding. Figure 5B is a Scatchard plot (36) of the same data. For this preparation the equilibrium dissociation constant KD was 0.16 nM, and the total number of specific binding sites for TCDD was 69 fmol/mg cytosolic protein. Similar results were obtained using MDEG buffer (25 mM MOPS, pH 7.4; 1 mM dithiothreitol; 1 mM EDTA; 5% glycerol) or KTMD buffer (100 mM KCI; 25 mM Tris-HCl, pH 7.4; 5 mM MgCl,; 1 mM dithiothreitol). Table 1 shows the results of analysis of the hepatic cytosolic receptor in Sprague-
RECEPTORS
7
Dawley rats, as well as C57BL/6J, DBA/ 23, and B6D2FI/J mice. In these experiments E3H]TCDD concentrations of 0.1 to 1.0 nM were used, and with the exception of hepatic cytosol from DBA/2J mice, the specific binding was greater than 4 times that of the nonspecific binding. The cytosol from rat liver contained binding sites with the highest affinity, 0.12 nM. Some apparent seasonal variation in the number of specific binding sites was observed in C57BL/6J mice. Seasonal variations in mouse hepatic cytosol receptor number also were reported by Okey et al. (28). Little specific binding was observed using the hepatic cytosol from DBA/U (Table 1, Fig. 6). With this strain of mice, the specific binding was 20-40s of the nonspecific binding and was considered too low to accurately quantitate the number of receptors or affinity. The hepatic cytosol from the B6D2FI/J cross showed somewhat intermediate specific binding as compared to the C57BL/6J or the DBA/2J parent strains (Table 1, Fig. 6).
13H] TCDD
(dpm x 10 3/0.2ml)
FIG. 6. Specific binding of [ ‘HJTCDD to the hepatic cytosol from C57BL/6J, DBA/2J, and B6D2F1/J mice. Specitic binding was determined by the HAP assay as described under Materials and Methods. The protein concentration for C57BL/6J mice (obtained FebruaryApril) was 1.70 mg/ml, for DBA/ZJ mice was 2.25 mg/ ml, and for B6D2F,/J mice (obtained February-April) was 2.10 mg/ml. Each point represents the mean of two determinations using the same cytosol preparation.
8
GASIEWICZ
AND NEAL
8 100 3%
90 80
5
70
8
60
5
50
g 2
do 30
iii g
20 10
s
lo-"
1O-9 CONCENTF3ATlON
Ida
id7 OF COMPETITOR
(M)
FIG. 7. Competition by dibenzo-p-dioxin congeners and other compounds for the specific binding of [3H]TCDD to rat hepatic cytosoi. The specific binding of 13H]TCDD to rat hepatic cytoso1 (2.15 mg protein/mi) was determined by the HAP assay as described under Materials and Methods. The concentration of [“HITCDD was 0.30 nM. The concentrations of other compounds is given on the abscissa.
Competitive binding by congeners of TCDD and other compounds. Figure 7 demonstrates that the specific binding of 13H]TCDD to rat hepatic cytosol as determined by the HAP assay is competitively inhibited by other compounds known to induce a pattern of monooxygenase activities which is similar to those induced by TCDD. The results are similar to those obtained previously using hepatic cytosol from C57BLf6J mice (14). As expected, the apparent binding affinities of 1,2,4-trichlorodibenzo-p-dioxin and dibenzo-p-dioxin for the rat hepatic cytosol receptor are 2.5 to 4 orders of magnitude lower than TCDD or TCDF. Phenobarbital, which induces a different pattern of microsomal monooxygenases from those induced by TCDD (6), shows no competitive binding at concentrations up to 1 X lo+ M. DISCUSSION
The results of these studies have shown that the HAP assay gives reliable and reproducible estimates of the number of specific TCDD binding sites in mouse and rat hepatic cytosol and their relative affinities for TCDD and related compounds. A higher ratio of specific to nonspecific binding is the
major advantage of this assay system over the previously published dextran-charcoal method (14), although the results obtained by the two methods are quite similar. The present method is also faster and appears to give more consistent data than the use of sucrose gradient techniques (28,29). The present method appears to have similar advantages over the isoelectric focusing procedure (27), which also requires trypsin treatment of the cytosol to prevent receptor aggregation. Utilizing the isoelectric focusing technique, Carlstedt-Duke and coworkers (27,39) reported concentrations of the receptor in hepatic cytosol from Sprague-Dawley rats of 14 to 31 fmol/mg protein with a Kn of 0.61 nM. These investigators incubated the cytosol with [3H]TCDD or [3H]TCDD + TCDF for 1 to 2 h at 0°C. Using the HAP assay under the same conditions, receptor concentrations of 20 to 30 fmol/mg protein were observed with Kn’s ranging from 0.2 to 0.8 nrvr. Additional experiments indicated that, at O”C, equilibration of the receptor in rat hepatic cytosol with added [‘H]TCDD or TCDF does not occur until 12 to 24 h (Fig. 4). Similar results were obtained using cytosol prepared from rat lung and thymus,
ASSAY FOR 2,3,7,8-TETRACWLORODIBENZO-P-DIOXIN
as well as hepatic cytosol from C57BL/6J mice. Thus, it is possible that, due to incomplete equilibration of the receptor with [ 3H]TCDD, the investigations by CarlstedtDuke and co-workers (27,39) may have underestimated the number of receptors and their relative affinity for TCDD. However, it should be pointed out that the times to equilibration observed in our laboratory differ considerably from those previously obtained by other investigators. Using a dextran-charcoal assay, Poland et al. (14) reported that constant specific binding is reached by 1 h at 0°C. Utilizing sucrose gradient techniques, Okey et al. (28) reported nearly complete equilibration within 5 min at 4°C. The reasons for these differences are as yet unknown. Hepatic cytosol from both rats and mice appear to reach constant specific binding rapidly at 20°C. In the present study, similar results for the number of specific binding sites and Kn values were obtained when the same rat hepatic cytosol preparation was incubated with various concentrations of E3H]TCDD and [3H]TCDD + TCDF for 2 h at 20°C or 24 h at 0°C. Hannah et al. (29) reported the Ah receptor in C57BLf6J mice to have an approximate molecular weight of 245,000. Using high-resolution gel filtration, we have determined the molecular weight of the specific binding peak in rat hepatic cytosol to be approximately 280,000.4 The specific binding observed by this gel-filtration method corresponds to the specific binding as determined by the assay presently described utilizing HAP. These preliminary studies suggest, but no not prove, that the specific binding as determined by the HAP assay is qualitatively comparable to that determined by other assay systems. Utilizing a sucrose gradient technique, Okey et al. (28) reported a Kn of 0.7 nM for TCDD specific binding sites in hepatic cytosol from C57BL/6J mice. Examination of 4 Gasiewicz, T. A., and Rucci, G., in preparation.
RECEPTORS
9
t3H]TCDD from the same supplier using high-~rformance liquid chromatography (33) revealed the [ 3H]TCDD was only about 65% pure. The main contaminating radioactive peaks had the same retention time as the tri- and pentachlorodibenzo-p-dioxin isomers. Thus, it is possible that the higher Kn value obtained by Okey et al. (28) may be in part a result of the presence of radiolabeled dioxin contaminants having a lower afiinity for the receptor. The previous investigations by Poland et al. ( 14) and Okey et ai. (28) reported either very low or unmeasurable specific binding of [ 3H]TCDD to hepatic cytosol from DBA/ 25 (nonresponsive) mice. Similar results were obtained in the present study. At present, adequate analysis of the specific binding in the DBA/U mouse hepatic cytosol cannot be performed due to the limited solubility as well as to the specific activity of the available E3H]TCDD. It is of interest that in hepatic cytosol preparations from B6D2 F,/J mice (offspring of C57BL/6J and DBA/2J parents, heterozygous at the Ah locus), the total amount of specific binding (Table 1, Fig. 6) is somewhat intermediate between that of the C57BL/6J and DBA/2J mice. However, the relative affinity for TCDD is only slightly different from that observed in C57BL/6J hepatic cytosol (Table 1). Intermediate responses of the B6D2FI/J offspring to TCDD have been observed for AHH induction (40), cleft palate production (15), thymic atrophy (151, and lethality.’ Although indirect evidence ( 14,16) does suggest that the major defect in the DBA/U mice is an altered receptor with reduced affinity toward TCDD, from the present data it is not possible to determine whether the low amount of specific binding observed in DBA/2.J mouse is due to a decrease in the number of receptor molecules, a decreased affinity, or a combination of these factors. It is also possible that the induction of AHH ’ Gasiewicz, T. A., Geiger, L. E., and Neal, R. A., in preparation.
10
GASIEWICZ
activity in nonresponsive mice by TCDD is initiated by a mechanism different than binding to this receptor protein in the cytoso1. Com~tition studies (Fig. 7) suggest that the specificity of binding of various compounds to the receptor species in SpragueDawley rats is quantitatively similar to that observed in the hepatic cytosol from C57BL/ 65 mice (14). These data support but are not suf?icient evidence for the hypothesis that, as in mice ( 14), the specific cytosolic binding of TCDD to the receptor in rats mediates AHH induction. Additional support comes from the observation that the ability of congeners of TCDD and other com~unds such as 3-methylcholanthrene and benzo[cu]pyrene to induce AHH activity is similar in both rats and mice (40-43). ACKNOWLEDGMENTS The excellent technical assistance of George Rucci is greatly appreciated. This study was supported in part by Grants ES-02515, ES-00267, ES-015.52, and ES05100 and Center Grant ES-01247 from the National Institute of Environmental Health Sciences, BRSG Grant RR 05403, and a grant from the Pharmaceutical Manufactures Association Foundation. This paper is also based on work partially performed under Contract DE-AC02-76EV03490 with the U. S. Department of Energy at the University of Rochester Department of Radiation Biology and Biophysics and has been assigned Report No. UR-3490-2035.
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McConnell, E. E., and Moore, J. A. (1979) Ann. N. I’. Acad. Sci. 320, 138-150. 5. Poland, A., Greenlee, W. F., and Kende, A. S. (1979) Ann. N. Y. Acad. Sci. 320, 214-230. 6. Nebert, D. W., and Jensen, N. M. (1979) CRC Act. Rev. Biochem. 6,401-437. 7. Negishi, M., and Nebert, D. W. (1979) J. Biol. Chem. 254, 11,015-l 1,023. 4.
AND NEAL 8. Owens, I. S. (1977)J. Biol. Chem. 252,2827-2833. 9. Kumaki, K., Jensen, H. M., Shire, J. 0. M., and Nebert, D. W. (1977) J. Biol. Chem. 252, 157165. IO. Nebert, D. W., Jensen, N. M., Perry, J. W., and Oka, T. (1980) J. Biol. Chem. 255,6836-6842. 11. Gielen, J.E., Goujon, F. M., and Nebert, D. W. (1972) J. Biol. Chem. 247, 1125-l 137. 12. Nebert, D. W., and Gielen, J. E. (1972) Fed. Proc. 31, 1315-1325. 13. Thomas, P. E., Kouri, R. E., and Hutton, J. J. (1972) Biochem. Genet. 6, 157-168. 14. Poland, A., Glover, E., and Kende, A. S. f 1976) J. Biol. Chem. 251,4936-4946. 15. Poland, A., and Clover, E. (1980) Mol. Pharmacol. 17, 86-94.
16. Greenlee, W. F., and Poland, A. (1979) J. Biof. Chem. 254,9814-9821. 17. Kouri, R. E., and Nebert, D. W. (1977) in Origins of Human Cancer (Watson, J. D., and Hiatt, H., eds.), pp. 81 l-835, Cold Spring Laboratory, New York. 18. Kouri, R. E., Ratrie, H., and Whitmire, C. E. (1973) J. Nat. Cancer inst. 51, 197-200. 19. Kociba, R. J., Keyes, D. G., Beyer, J. E., Carreon, R. H., Wade, C. E., Dittenber, D. A., Kalnins, R. P., Frauson, L. E., Park, C. N., Barnard, S. D., Hummel, R. A., and Humiston, C. G. (1978) Toxicol. Appl. Pharmacol. 46,279-303. 20. Toth, R., Sugar, J., Somfai-Felle, S., and Bence, J. (1978) Progr. Biochem. Pharmacof. 14, 8293. 21. VanMiller, J. P., Lalich, J. J., and Allen, J. R. (1977) Chemosphere 6,537-544. 22. Poland, A., and Glover, E. (1979) Cancer Res. 39, 3341-3344. 23. Geiger, L. E., and Neal, R. A. (1981) Toxicol. Appl. Pharmacol. 59, 125-l 29. 24. Nebert, D. W., Thorgeirsson, S. S., and Felton, J. S. (1976) in In Vitro Metabolic Activation in Mutagenesis Testing (DeSerres, F. J., Fouts, J. R., Bend, J. R., and Philpot, R. M., eds.), pp. 105-124, Elsevier-North-Holland, Amsterdam/ New York. 25. Wassom, J. S., Huff, J. E., and Laprieno, N. ( 1978) Mutat. Res. 47, 141-160. 26. Pitot, H. C., Goldsworthy, T., Campbell, H. A., and Poland, A. (1980) Cancer Res. 40, 3616-3620. 27. Carlstedt-Duke, J., Elfstrom, G., Snochowski, M., Hogberg, B., and Gustafsson, J. (1978) Toxicol. Lett. 2, 365-373. 28. Okey, A. B., Bondy, G. P., Mason, T. M., and Nebert, D. W. (1979) J. Biol. Chem. 254, 11,636-l 29.
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37. Gorski, J., and Gannon, F. (1976) Annu. Rev. Physiol. 38,425-450. 38. Rousseau, G. G., Higgins, S. J., Baxter, J. D., Gelfand, D., and Tomkins, G. M. (1975) .I. Biol. Chem. 250,6015-6021. 39. Carlstedt-Duke, J. M. B. (1979) Cancer Res. 39, 3172-3176. 40. Poland, A., and Glover, E. (1975) Mol. Pharmacol. 11, 389-398. 4 1. Poland, A. P., and Glover, E. (1973) Mol. Pharmacol. 9, 736-747. 42. Poland, A. P., and Glover, E. (1974) Mol. Pharmacol. 10, 349-359. 43. Poland, A. P., Glover, E., Robinson, J. R., and Nebert, D. W. (1974) J. Biol. Chem. 249,55995606.