Effects of ligand structure on the in vitro transformation of the rat cytosolic aryl hydrocarbon receptor

ARCHIVES

OF BIOCHEMISTRY

AND

BIOPHYSICS

Vol. 297, No. 1, August 15, pp. 73-79, 1992

Effects of Ligand Structure on the in vitro Transformation of the Rat Cytosolic Aryl Hydrocarbon Receptor’ M. Santostefano, Department

J. Piskorska-Pliszczynska,2

of Physiology and Pharmacology,

V. Morrison,

Texas A&M

University,

and S. Safe3 College Station, Texas 77843-4466

Received March 2, 1992, and in revised form April 17, 1992

Incubation of radiolabeled 2,3,7&tetrachlorodibenzop-dioxin (TCDD), 2,3,7,Stetrachlorodibenzofuran (TCDF),1,2,3,7,8-pentachlorodibenzo-p-dioxin(PeCDD), 1,2,3,7&pentachlorodibenzofuran (PeCDF), 1,2,7,8TCDF, and 2,3,7-trichlorodibenzo-p-dioxin (TrCDD) with rat hepatic cytosol for 2 h at 0°C gave liganded aryl hydrocarbon (Ah) receptor complexes which were indistinguishable as determined by velocity sedimentation analysis and DNA-Sepharose column chromatography. Incubation of the cytosol plus the different radioligands for 2 h at 20°C resulted in the formation of Ah receptor complexes which exhibited increased retention times on DNA-Sepharose columns. It was apparent that the amount of specifically bound Ah receptor complex or the levels of the transformed Ah receptor complex which eluted from the column with 0.2-0.3 M salt were dependent on the structure of the radioligand. For example, after incubation for 2 h at 20°C the overall yields of the specifically bound transformed Ah receptor complex were 3.4,2.0,1.2,1.9, 0.3,andO.l%,respectively,using2,3,7,8-TCDD,2,3,7,8TCDF, 1,2,3,7,6PeCDD, 1,2,3,7,8-PeCDF, 1,2,7,8TCDF, and 2,3,7-TrCDD as radioligands. A more quantitative measure of the structure-dependent transformation of the liganded cytosolic Ah receptor complex was determined using a gel retardation assay with a consensus synthetic dioxin-responsive element (DRE) (26-mer, duplex). The EC,, values obtained for the concentration-dependent formation of the retarded DRE-Ah receptor complexusing2,3,7,8-TCDD, 1,2,3,7,8-PeCDD,2,3,7,8TCDF, 1,2,3,7,8-PeCDF, 2,3,7-TrCDD, and 1,2,7,8TCDF as ligands were 0.26, 0.35, 0.78, 1.75, 27.0, and The structure-dependent differences 220 nM, respectively. in these values were similar to their different potencies * This work was supported by a grant from the National Institutes of Health (ES03554 and P42-ES04917) and the Texas Agricultural Experiment Station. ’ On leave from the Veterinary Research Institute, Pulawy, Poland. 3 To whom correspondence should be addressed. 0003.9861/92 $5.00 Copyright 0 1992 by Academic Press, All rights of reproduction in any form

as Ah receptor agonists and these data suggest that the structure-dependent transformation of the liganded cytosolic Ah receptor may significantly contribute to the structure-activity relationships observed for 2,3,7,8TCDD and related compounds. o 1992 academic press, IUC.

The aryl hydrocarbon (Ah)4 receptor is a widely distributed intracellular protein which binds saturably and with high affinity to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), 3-methylcholanthrene (MC), and structurally related aromatic and chlorinated aromatic hydrocarbons (l-11). No endogenous ligand for the Ah receptor has been identified; however, indolo[3,2-blcarbazole, which is formed from the acid-catalyzed conversion of the natural secondary plant metabolite indole-3-carbinol, binds with moderately high affinity to the Ah receptor (12,13). The molecular mechanisms of Ah receptor-mediated responses have been derived from studies on the molecular biology of MC- and TCDD-induced CYPlAl gene expression (1423). The results indicate that the liganded nuclear Ah receptor acts as a nuclear ligand-responsive transcriptional factor (LTF) (24) and gene transcription is induced only after interaction with sequence-specific genomic dioxin-responsive elements (DREs) which are located in the 5’-region flanking the CYPlAl gene. The formation of the nuclear Ah receptor is the result of a sequence of events which exhibit similarities and differences with the steroid and thyroid hormone family of receptors (24-27). The unbound Ah receptor appears to 4 Abbreviations used: Ah, aryl hydrocarbon; DRE, dioxin-responsive element; Hsp, heat shock protein; LTF, ligand-responsive transcriptional factor; MC, 3-methylcholanthrene; PCDD, polychlorinated dibenzo-pdioxin; PCDF, polychlorinated dibenzofuran; PeCDD, pentachlorodibenzo-p-dioxin; PeCDF, pentachlorodibenzofuran; TCDD, tetrachlorodibenzo-p-dioxin; TCDF, tetrachlorodibenzofuran; TrCDD, trichlorodibenzo-p-dioxin; DMSO, dimethyl sulfoxide. 73

Inc. reserved.

74

SANTOSTEFANO

be a multimeric cytosolic complex which is associated with heat shock protein (Hsp) 90 (28). After initial binding to a ligand, such as TCDD, the 9-10 S complex undergoes transformation (29-33) to form a heterodimer which contains the liganded Ah receptor and the Arnt protein (3436). This latter protein confers increased DNA binding activity on the cytosolic Ah receptor complex which readily undergoes translocation into the nucleus. The transformed cytosolic Ah receptor complexes from different species also exhibit several common properties, including a decreased molecular weight due to the loss of Hsp 90, increased binding affinity on DNA-Sepharose columns, and the sequence-specific binding to DNA (i.e., DREs) as determined by gel shift assays (25-27, 37-41). The transformed cytosolic and nuclear Ah receptor complexes appear to be identical heterodimers (42) which contain a ligand binding (Ah receptor) subunit and the Arnt protein which is important for nuclear translocation activity (24-27). The results from in vitro studies indicate that the rate of transformation of the cytosolic receptor is dependent on both species and organ sources of the receptor preparation (27, 33). One of the hallmarks of the Ah receptor-mediated responses is the correlation between the structure-receptor binding versus structure-activity relationships for both aromatic and chlorinated aromatic hydrocarbons (43). Thus, for a series of compounds, such as TCDD and structurally related polychlorinated dibenzo-p-dioxin and dibenzofuran congeners, their rank order competitive binding affinities were comparable to their potencies as inducers of CYPlAl gene expression or other Ah receptormediated biochemical and toxic responses. These data suggest that the major structure-dependent step in the conversion of the unoccupied Ah receptor to the transformed nuclear Ah receptor complex is associated with the initial binding of the ligand to the cytosolic Ah receptor. However, there is also evidence to suggest that the initial ligand-receptor interaction may not be the only structure-dependent step in this complex process. For example, several ligands such as the 6-alkyl-1,3&trichlorodibenzofurans exhibit relatively high competitive binding affinities but low Ah receptor agonist activities (44, 45); the Scatchard-derived KD values for several radiolabeled PCDD and PCDF congeners were not structuredependent (3, 46); and using protein dilution and kinetic techniques the KD values for the binding of TCDD to the Ah receptor were two to three orders of magnitude lower than those derived by standard Scatchard analysis (47, 48). Therefore the major objectives of this project were to examine the temperature-dependent transformation of the rat cytosolic Ah receptor liganded with 2,3,7,8-TCDD, 1,2,3,7,8-pentachlorodibenzo-p-dioxin (PeCDD), 1,2,3,7,8pentachlorodibenzofuran (PeCDF), 2,3,7,&tetrachlorodibenzofuran (TCDF), 2,3,7-trichlorodibenzo-p-dioxin (TrCDD), and 1,2,7,8-TCDF using DNA-Sepharose column chromatography and gel retardation assays. This

ET AL.

approach should delineate the effects of ligand structure on the formation of the transformed cytosolic Ah receptor. MATERIALS

AND METHODS

Chemicals and biochemicals. The radiolabeled polychlorinated dibenzo-p-dioxin and dibenzofuran congeners were synthesized via the microchlorination of [1,6-3H2]dibenzo-p-dioxin and [1,4,63H&libenzofuran as described (10). The radiolabeled 2,3,7,8-[3H]TCDD (37 Ci/mmol), 2,3,7,8-[3H]TCDF (57 Ci/mmol), 1,2,3,7,8-[3H]PeCDF (34 Ci/mmol), 1,2,3,7,8-[3H]PeCDD (29 Ci/mmol), l,2,7,8-[3H]TCDF (56 Ci/mmol), and 2,3,7-[3H]TrCDD (37 Ci/mmol) were 195% pure as determined by gas chromatographic analysis using electron-capture detection. The unlabeled congeners were synthesized in this laboratory and were >95% pure as determined by gas chromatographic analysis using electron-capture detection. The unlabeled congeners were synthesized in this laboratory and were >98% pure (38). Hydroxylapatite was purchased from Bio-Rad Laboratories (La Jolla, CA). A complementary pair of synthetic oligonucleotides containing the sequence 5’. GATCTGGCTCTTCTCACGCAACTCCG-3’ was synthesized, purified by polyacrylamide gel electrophoresis, and annealed as described (44). The oligonucleotide was labeled at the 5’ end using T4-polynucleotide kinase and [y-32P]ATP purchased from New England Nuclear (Boston, MA). Calf thymus DNA and N-ethyl-IV’-[3-(dimethylamino)propyl]carbodiimide hydrochloride were purchased from Sigma Chemical Co.; CH Sepharose 4B powder was obtained from Pharmacia, Liquiscent from National Diagnostic. All other chemicals were analytical grade products from either Sigma or Fisher Chemical Co. Animals. Male Long Evans rats (21 days old; = 100 g) were obtained from Harlan Laboratories (Houston, TX) and were housed in plastic cages with hardwood bedding, allowed free access to Purina certified rodent chow, No. 5002, and water, and maintained on a diurnal cycle of 12 h of light and 12 h of darkness. Preparation of cytosol. The animals were terminated by cervical dislocation and the livers were immediately perfused in situ by the hepatic portal vein with ice-cold HEGD buffer [25 nM Hepes, 1.5 mM EDTA, 1 mM dithiothreitol, 10% glycerol (v/v), pH 7.61. The excised livers were rinsed once with 10 ml of the fresh ice-cold HEGD buffer and finely minced. The minced livers were rinsed again with buffer and homogenized in ice-cold HEGD buffer using a Brinkman PT45/80 homogenizer. The homogenate was centrifuged at 10,OOOgfor 20 min at 2°C and the surface lipids were removed by aspiration. The resulting supernatant was recentrifuged at 105,OOOgfor 1 h at 2°C and again the surface lipids were removed. Protein concentrations were measured by the method of Bradford (49) and the supernatant (cytosol) was stored at -80°C until used. In vitro transformation with different rodioligands. Radioligands were added to cytosol (2 mg protein/ml) in DMSO (10 PI/ml of cytosol) at a final concentration of 10 nM with or without 200-fold excess of unlabeled 2,3,7,8-TCDF. Cytosols were incubated for 2 h at 4°C or 2 h at 20°C for transformation. Free and loosely bound ligands were removed by a dextran-coated charcoal pellet (10 mg charcoal and 1 mg of dextran per 1 ml of incubation mixture) and after centrifugation the cytosols were used for column chromatography. DNA-Sepharose column chromatography. DNA-Sepharose was prepared as described (41) using washed CH Sepharose (CB) (Pharmacia, Sweden). The DNA coupling procedure gave yields of 0.8 mg/ml of swollen gel. Three milliliters of labeled crude cytosol samples was applied onto the column, which was eluted with 40 ml HEDG buffer and then subsequently with a 60-ml linear gradient consisting of O-O.5 M sodium chloride in HEDG buffer. Fractions of 1 ml were collected and measured for conductivity and radioactivity. Hydroxylapatite aasoy. The E&s values for competitive receptor binding affinities were determined using freshly prepared rat hepatic cytosol (2 mg protein/ml) and the hydroxylapatite assay procedure essentially as described (50). Different concentrations of 2,3,7,8-TCDD,

EFFECTS

OF LIGAND

STRUCTURE

ON THE

RAT

1,2,7,8-TCDF, 2,3,7&TCDF, 1, 2 t3 97 78-PeCDF, 1 ,2 73 , 7 78-PeCDD, and 2,3,7-TrCDD were used to determine the displacement curves; the EC$,,, values were defined as the concentrations required to displace 50% of the 2,3,7,8-[3H]TCDD and were determined graphically from a log-logit plot of the percentage of 2,3,7,8-t3H]TCDD bound versus log concentration of the ligand. Gel retardation analysis (37). Complementary strands of the synthetic oligonucleotide containing a consensus DRE sequence (26-mer) were synthesized, purified by polyacrylamide gel electrophoresis, and annealed. The oligonucleotide was labeled at the 5’ end using T4-polynucleotide kinase and [y-“‘P]ATP. Cytosol (8 mg protein/ml) was incubated with DMSO (20 pi/ml) and various concentrations of 2,3,7,8-TCDD, 1,2,7,8TCDF, 2,3,7,8-TCDF, 1,2,3,7,8-PeCDF, 1?2 >3 97 ,8-PeCDD, and 2,3,7TrCDD ranging from 1O-7 to 10-l’ M for 2 h at 20°C prior to gel retardation analysis. Cytosol (40 Ng) was incubated in HEDGK (HEGD + for 15 min at 20°C to bind nonspecific 0.8 M KCl) with 200 ngpoly(dI-C) DNA-binding proteins. Following the addition of 100,000 cpm (0.2-0.8 ng) of [32P]DRE oligonucleotides, the reaction mixtures were incubated for 15 min at 25°C. Protein-DNA complexes were resolved on 5% polyacrylamide gel (acrylamide:bisacrylamide ratio of 3O:O.a) at 120 V using a Tris borate-EDTA buffer. Gels were dried and protein-DNA binding was visualized by autoradiography. To determine the amount of proteinDNA complexes formed, the specific radiolabeled band was quantitated using a Betascope 603 blot analyzer. The amount of [32P]DRE specifically bound in the ligand-inducible complex was estimated by measuring the amount of radioactivity in the inducible protein-DNA complex, isolated from a ligand-treated sample lane, and subtracting the amount of radioactivity present in the same position in a non-ligand-treated lane. The difference in radioactivity between these samples represents the ligand-inducible specific binding of the Ah receptor complex to the [32P]DRE. The levels of DNA-binding activity for the various PCDDs/ PCDFs congeners were quantitated as a percentage of the maximal response observed in the TCDD-treated cytosol. The EC, values were obtained from a log-log& plot of percentage of maximal response obtained for each ligand versus ligand concentration. Each data point for the DRE binding studies is expressed as a mean + SE for at least three separate determinations.

RESULTS

The ligands selected for this study were a series of four 2,3,7,8-substituted congeners, namely 2,3,7,8-TCDD, 2,3,7,8-TCDF, 12, 73 97 ,8-PeCDD, and 12, ,3 77 ,8-PeCDF, and two compounds with only three lateral chlorine substituents, 2,3,7,-TrCDD and 1,2,7,8-TCDF. The competitive displacement binding affinities of the six congeners using 2,3,7,8-[3H]TCDD as the radioligand were determined using Long Evans rat hepatic cytosol and the hydroxylapatite binding assay procedure (Fig. 1). Logit analysis of the competitive binding data (four separate determinations per compound) gave EC& values of 1.78 -t 1.14, 1.01 f 0.46, 1.34 f 1.39, 0.67 k 0.30, 4.34 + 0.43, and 9.68 +- 4.32 nM for 2,3,7,8-TCDD, 2,3,7,8-TCDF, 2,3,7-TrCDD, and 1>2 93 T7 98-PeCDD, 1,2,3,7,8-PeCDF, 1,2,7,8-TCDF, respectively. The EC,, values for the 2,3,7,8-substituted compounds were not significantly different, whereas the EC&‘s for the 2,3,7-TrCDD and 1,2,7,8-TCDF were significantly higher (P < 0.05). Incubation of radiolabeled 2,3,7,8-TCDD for 2 hat 4°C followed by chromatography of an aliquot of this sample on a DNA-Sepharose column eluted with a salt gradient gave an elution profile which is illustrated in Fig. 2 (top). A single peak was eluted at a salt concentration of 0.05

CYTOSOLIC

ARYL

.Ol

HYDROCARBON

.l

1 [Ligand]

RECEPTOR

10

100

1000

(ribI)

FIG. 1. Competitive displacement of 2,3,7,8-[3H]TCDD from the rat hepatic cytosolic receptor by unlabeled PCDD and PCDF ligands. Hepatic cytosol (2 mg protein/ml) was incubated with 2,3,7,8-[3H]TCDD and different concentrations of unlabeled competitors, and the levels of specific binding were determined by the hydroxylapatite binding assay as described under Materials and Methods. The EC& values were determined from log-logit plots of the data. The binding curves were determined four times for each ligand and the EC& values for 2,3,7,8TCDD (W), 2,3,7,8-TCDF (A), 1,2,3,7,8-PeCDD (a), 1,2,3,7,8-PeCDF (0), 2,3,7-TCDD (O), and 1,2,7,8-TCDF (0) were 1.78 f 1.14, 1.01 + 0.46,1.34 + 1.39,0.64 + 0.30,4.34 f 0.43, and 9.68 + 4.32 nM, respectively.

and velocity sedimentation analysis of this fraction gave a specifically bound peak which sedimented at 9.6 S (data not shown). In a parallel experiment, the radioligand and the rat hepatic cytosol were incubated for 2 h at 20°C and the elution profile of this incubation mixture on a DNASepharose column is illustrated in Fig. 2 (bottom). The specifically bound untransformed cytosolic Ah receptor complex was not detected in the fractions eluted with salt gradient from 0 to 0.1 M, whereas two additional specifically bound peaks were observed in fractions eluted with 0.1-0.2 and >0.2 M salt buffer. The latter peak which eluted with a salt concentration of 0.25 M KC1 was identical to the transformed or nuclear Ah receptor complex and velocity sedimentation analysis gave a specifically bound peak which sedimented at 6.8 S. The two peaks associated with the transformed receptor were also observed as minor peaks after incubation of the cytosol for 2 h at 4°C (Fig. 2, top, insert). Incubation of radiolabeled 2,3,7,8-TCDF, 1,2,3,7,8-PeCDD, 1,2,3,7,8-PeCDF, 2,3,7TrCDD, and 1,2,7,8-TCDF for 2 h at 0°C with rat hepatic cytosol followed by charcoal treatment and DNA-Sepharose column chromatography using salt gradient elution gave a specifically bound peak which eluted from the column as illustrated in Fig. 2 (top). In a parallel experiment in which the radioligands were incubated for 2 h at 2O”C, their elution profiles from a DNA-Sepharose column were dependent on radioligand structure. In all cases, there was an increased elution of specifically bound peaks in the fractions containing 0.1-0.2 and >0.2 M salt and this resembled, in part, the results obtained for 2,3,7,8M

76

SANTOSTEFANO DNAaffinity chromatography

Long Evans Rat cytosol

RACTION NUMBER (ml) milliMH0 ) [ 0

p.1010.20 IO.32 N&l ( M )

FIG. 2. DNA-Sepharose affinity chromatography of the cytosolic 2,3,7,8-[3H]TCDD Ah receptor complex for 2 h at 4°C (top) or 2 h at 20°C (bottom). The cytosols were incubated with either 2,3,7,8[sH]TCDD (Cl) or 2,3,7,8-[3H]TCDD plus a 200-fold molar excess of unlabeled 2,3,7,8-TCDF (*). Aliquots of the cytosolic receptor complex were layered on the DNA-Sepharose columns and eluted with 60 ml of a O-O.5 M KC1 gradient as described (41). The major specifically bound peak which was formed after incubation for 2 h at 4’C eluted with a salt concentration of 0.05 M KC1 (top); the major specifically bound peaks which formed after incubation for 2 h at 20°C eluted with salt concentrations of 0.15 and 0.25 M KCl. The insert (top) is plotted on the same scale as the elution profile of the transformed receptor complex (bottom). The relatively low concentrations of the minor peaks were eluted with salt concentrations of 0.15 and 0.25 M KC1 and corresponded to the values obtained for the major peaks eluted from the transformed rat cytosol preparation (bottom).

t3H]TCDD (Fig. 2, bottom). However, there was considerable radioligand-dependent variability in the shapes of the eluted peaks and their relative distribution in the fractions eluted with O-0.1, 0.1-0.2, and >0.2 salt. It was also apparent that there was a marked increase in the radioactivity eluted in the void volume after incubation of hepatic cytosol with radiolabeled 1,2,7,8-TCDD and 2,3,7-TrCDD for 2 h at 20°C. The results in Table I summarize the data obtained using a single rat hepatic cytosol preparation in which the amount of specifically bound transformed receptor which eluted with >0.2 M salt from a DNA-Sepharose column was quantitated. The results showed that the total amount of specifically bound radioactivity which formed after incubation of the cytosol plus the different radioligands for 2 h at 20°C was 13.9, 8.0, 14.5, 15.0, 3.9, and 3.2% for 2,3,7,8-TCDD, 2,3,7,8TCDF, 1,2,3,7,8-PeCDF, 1,2,3,7,8-PeCDD, 1,2,7,8-TCDF,

ET AL.

and 2,3,7-TrCDD, respectively. The yields of specificallybound Ah receptor complex eluted from the column in fractions A-C or fraction C (see Fig. 2, bottom) expressed as a percentage of the total radioligand used or the amount applied to the column were structure-dependent with the lowest overall yields observed for the 1,2,7,8-TCDF and 2,3,7-TrCDD congeners which contain only the three lateral chlorine substituents. The ligand structure-dependent transformation of the rat cytosolic Ah receptor was also determined using unlabeled ligands followed by incubation with a consensus 32P-labeled DRE. The retarded band was determined by gel shift assays and quantitated using a Betagen Betascope 603 blot analyzer. The time-course ligand-dependent formation of the retarded DRE-protein complex is illustrated in Fig. 3. Both 2,3,7,8-TCDD and 2,3,7,8-TCDF formed the highest levels of retarded complex which maximized between 90 and 120 min and was relatively stable for up to 240 min. The lowest levels of DRE complex were observed for 1,2,7,8-TCDF and the curves for the other ligand-bound complex were observed for 1,2,7,8-TCDF and the curves for the other ligand-bound complex were intermediary between those obtained using 1,2,7,8-TCDF and 2,3,7,8-TCDD as receptor ligands. The ligand concentration-dependent formation of the retarded band was also determined using the gel shift assay procedure (Figs. 4 and 5). There was a concentration-dependent increase in the formation of the transformed receptor complex; the highest levels of the DRE-binding receptor complex were formed with the 2,3,7,8-substituted congeners, whereas lower concentrations were observed after incubation of the cytosols with 1,2,7,8-TCDF or 2,3,7-TrCDD. These results were comparable to those obtained using

TABLE

I

Radioligand-Dependent Transformation of the Rat Hepatic Cytosolic Ah Receptor after Incubation at 20°C for 2 h

Radioligand 2,3,7,8-TCDD 2,3,7,8-TCDF 1,2,3,7,8-PeCDD 1,2,3,7,8-PeCDF 1,2,7,8-TCDF 2,3,7-TrCDD

% Specifically bound” 13.9 8.0 14.5 15.0 3.9 3.2

% Eluted from DNA-Sepharose”sb 4.9 3.5 5.2 4.4 0.9 0.4

% Eluted in fraction C”” 3.4 2.0 1.2 1.9 0.3 0.1

(LDetermined as a percentage of the total amount of radioligand added to the incubation mixture. b These values were determined for the specifically bound radioactivity in fractions A, B, and C eluted from the DNA-Sepharose column (see Fig. 1. Note: A, O-0.1; B, 0.1-0.2; C, 0.2-0.3 M salt) c These values were determined only for the specifically bound fraction C which eluted from the DNA-Sepharose column at a salt concentration of 0.2-0.3 M (see Fig. 1).

EFFECTS

OF LIGAND

STRUCTURE

ON THE

RAT

60

0

100

Time

200

(min)

FIG. 3. Ligand-dependent time-course formation of the transformed cytosolic Ah receptor complex by gel shift assays. 2,3,7&TCDD (m), 2,3,7&TCDF (A), 1,2,3,7&PeCDD (A), 1,2,3,7&PeCDF (O), 2,3,7TrCDD (Cl), and 1,2,7&TCDF (0) (2 nM for all ligands) were incubated with rat hepatic cytosol for different time points and the DRE binding was determined by gel shift assays as described under Materials and Methods. Each was determined in triplicate and the results are expressed as means + SE. The maximum levels of DRE binding were obtained for 2,3,7&TCDD and the results are expressed as a percentage of the maximum response observed for this ligand.

CYTOSOLIC

ARYL

HYDROCARBON

RECEPTOR

77

degradation of unbound cytosolic Ah receptor (48) which are encountered in the in vitro binding assay. The effects of ligand structure on the formation of the Ah receptor complex was further investigated in this study by measuring the formation of the transformed cytosolic Ah receptor complex. The first approach utilized a series of radiolabeled PCDD and PCDF congeners (3, 46, 51) with high specific activities (29 to 57 Ci/mmol) which could be used as radioligands to determine the effects of ligand structure on the initial binding and subsequent transformation of the rat cytosolic Ah receptor after incubation for 2 h at 0 or 20°C. The results in Fig. 2 illustrate that the untransformed receptor using 2,3,7,8-[3H]TCDD as a radioligand was eluted from a DNA-Sepharose column with low salt (O-O.1 M), whereas, after transformation at 20°C for 2 h, only two specifically bound peaks which eluted at 0.1-0.2 and > 0.2 M salt were observed. The latter peak sedimented at 6-7 S (data not shown) and represented the transformed Ah receptor complex. The peak which eluted with a salt concentration between 0.1 and 0.2 M has previously been observed (25,33) and may be the Ah receptor (monomer) which is presumably a precursor of the transformed heterodimer. Incubation of the other 2,3,7,8-substituted radioligands with rat hepatic cytosol for 2 h at 0 or 20°C gave DNA-Sepharose column elution profiles which were similar to those obtained for 2,3,7,8-TCDD, although there were differences in the relative amounts of peaks A (O-O.1 M salt), B (0.1-0.2 M

DNA-Sepharose column chromatography; however, the gel shift assay procedure was more rapid and reproducible. DISCUSSION Rat cytosolic receptor is readily transformed by either temperature or high salt and was chosen as an appropriate model to investigate the effects of ligand structure on the transformation process. The ligands chosen for this study were all PCDD or PCDF congeners which exhibit Ah receptor agonist activity but differ markedly in their potency (i.e., 2,3,7,8-TCDD > 2,3,7,8-TCDF - 127 93 f7 98-PeCDD - 192 ,3 97 78-PeCDF > 2,3,7-TrCDD 2 1,2,7,8-TCDF) (38, 51). The results illustrated in Fig. 1 also show that the competitive binding affinities of the 2,3,7,8-substituted compounds to the cytosolic Ah receptor were not significantly different and were significantly lower than the corresponding ECbO values for the congeners with only three lateral substituents, namely 2,3,7-TrCDD (ECsO = 4.34 + 0.43 nM) and 1,2,7,8-TCDF (ECbO = 9.68 + 4.32 nM). However, it was also apparent that the approximate l&fold differences in competitive binding affinities were significantly lower than the > 45O-fold differences in their potencies as inducers of aryl hydrocarbon hydroxylase activities in rat hepatoma H-411 E cells in culture (51). The poor quantitative correlations between the structurebinding and structure-activity relationships are due, in part, to the high level of nonspecific binding (47) and

150

100

50

0 0.001

0.010

0.100

[Ligand]

FIG. 4. Concentration-dependent

1.000

10.000100.000

(nM)

formation of the transformed cytosolic Ah receptor complex as determined by gel shift assays. 2,3,7,8TCDD (W, 2,3,7&TCDF (A), 1,2,3,7&PeCDD (A), 1,2,3,7&PeCDF (O), 2,3,7-TrCDD (O), and 1,2,7&TCDF (0) (2 nM for all ligands) were incubated with rat hepatic cytosol at different concentrations and the DRE binding was determined by gel shift assays as described under Materials and Methods. Each data point was determined in triplicate and the results are expressed as means k SE. The maximum levels of DRE binding were assigned a value of 100% and the results for the other congeners were expressed as a percentage of this response.

78

SANTOSTEFANO

TCDD I

I

f-

AhR-DRE Complex

f-

Free DRE Probe

FIG. 5. Gel retardation of the transformed cytosolic Ah receptor complex treated with different concentrations of 2,3,7&TCDD. Different concentrations of 2,3,7&TCDD were added to hepatic cytosol, incubated for 2 h at 0°C and then incubated with synthetic DRE as described under Materials and Methods. The results illustrate the concentrationdependent formation of the retarded band (indicated with an arrow) which maximizes at lo-100 nM concentration. The radioactivity in the retarded band can be competitively displaced with increasing concentrations of unlabeled DRE (data not shown).

salt), and C (>0.2 M salt). In contrast, the corresponding elution patterns observed when 1,2,7,8-TCDF and 2,3,7TrCDD were used as radioligands were poorly resolved; there was a decrease in levels of specifically bound radioactivity eluted by the salt gradient and this corresponded to an increased amount of radioactivity eluted in the void volume. Thus, the two compounds with the lowest apparent competitive binding affinities, namely 2,3,7TrCDD and 1,2,7,8-TCDF, formed complexes which were relatively unstable after incubation at 20°C and did not readily undergo transformation. The results in Table I were obtained by incubating the radioligands with the same freshly prepared rat hepatic cytosol for 2 h at 20°C followed by DNA-Sepharose column chromatography. The results showed that the amount of specifically bound complex which eluted from the DNA-Sepharose column in 0.1-0.2 M or >0.2 M salt as a percentage of the initial amount of radioligand used was dependent on radioligand structure. For example, the percentage recovery of fully transformed cytosolic receptor (fraction C) liganded with the most toxic ligand, namely 2,3,7,8-TCDD, was 3.4% (based on the total amount of radioligand added to the incubation mixture). In contrast, the percentage recovery of transformed receptor for the other 2,3,7,8-substituted congeners varied between 1.2 and 2.0% and this decreased

ET AL.

recovery correlated with the decreased biochemical and toxic potencies of these compounds (51). 2,3,7-TrCDD and 1,2,7,8-TCDF are substituted with only three lateral chlorine groups and are significantly less active than the other four congeners used in this study. Not surprisingly the recovery of the transformed cytosolic receptor complexes liganded with 2,3,7-TrCDD and 1,2,7,8-TCDD was 0.1 and 0.3%, respectively, and this relatively poor yield of transformed Ah receptor corresponded to the decreased Ah receptor agonist activity of these congeners. Thus, the results of this study suggest that the structure-dependent transformation of the cytosolic Ah receptor was similar to the structure-dependent potencies of these congeners as Ah receptor agonists (43). Previous studies have demonstrated that the transformed and nuclear Ah receptor interact with a consensus [32P]DRE to form a retarded complex which can be readily detected and quantitated using a gel shift assay procedure (37-41,52-54) as illustrated in Fig. 5 using 2,3,7,8-TCDD as a ligand. This technique obviates the need for the difficult synthesis of the radioligands and provides an assay system which is more readily adapted for quantitative analysis. The results in Fig. 3 and 4 illustrate the timeand concentration-dependent formation of the DRE-receptor complexes after activation of the cytosol at 20°C in the presence of unlabeled ligands. Within 90-120 min, the maximum levels of DRE binding complex were formed with all the ligands. The lowest levels of complex formed in the time-course study were observed with 2,3,7-TrCDD and 1,2,7,8-TCDF, although surprisingly low levels were also observed for 1,2,3,7,8-PeCDF. In contrast, logit analysis of the concentration-dependent formation of the DRE complex gave ECsO values of 0.26, 0.35, 0.78, 1.75, 27.0, and 220 nM for 2,3,7,8-TCDD, 1,2,3,7,8PeCDD, 2,3,7,8-TCDF, 1,2,3,7,8-PeCDF, 2,3,7-TrCDD, and 1,2,7,8-TCDF, respectively. The differences in EC&,-, values were >840-fold and approximated the relative potencies of these compounds as Ah receptor agonists (43, 51). The results of this in vitro study show that the formation of the transformed rat cytosolic Ah receptor complex is ligand structure-dependent for selected PCDD and PCDF congeners. Comparable results have also been reported for polynuclear aromatic hydrocarbons which are Ah receptor agonists (41). Moreover, for the PCDD and PCDF congeners, the structure-dependent formation of the transformed cytosolic Ah receptor complex corresponded to the structure-dependent accumulation of the nuclear Ah receptor complex in C67BL/6 mouse liver and rat hepatoma H-411 E cells treated with the same set of congeners (46,51). Therefore, these data suggest that the familiar structure-activity relationships observed for PCDDs and PCDFs (43) is due to the ligand structuredependent formation of the transformed cytosolic Ah receptor complex.

EFFECTS

OF LIGAND

STRUCTURE

ON THE

RAT

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