carcinogen-binding proteins in the rat ventral - cancer research

[CANCER RESEARCH 46, 651-657, February 1986]

Carcinogen-binding Proteins in the Rat Ventral Prostate: Specific

and Nonspecific High-Affinity Binding Sites for Benzo(a)pyrene,3-Methylcholanthrene, and 2,3,7,8-Tetrachlorodibenzo-p-dioxin1

Peter Soderkvist,2 Lorenz Poellinger, and Jan-Ã‚ke Gustafsson

Department of Medical Nutrition, Karolinska Institutet, Huddinge University Hospital F-69, S-141 86 Huddinge, Sweden

ABSTRACT

The polychlorinated dibenzodioxin [3H]-2,3,7,8-tetrachlorodi-benzo-p-dioxin (TCDD) and the carcinogens [3H]benzo(a)pyreneand [3H]-3-methylcholanthrene bound to saturable binding sites

in cytosol from the rat ventral prostate. Analysis of equilibriumbinding parameters in diluted cytosol preparations indicated anapparent Ka of ~2 nw and a binding capacity of approximately 1nmol/mg cytosolic protein, corresponding to ~5% of the total

protein content. However, gel permeation chromatography analysis as well as velocity sedimentation analysis on sucrose gradients of [3H]TCDD-labeled rat prostatic cytosol indicated bindingof [3H]TCDD to two discrete species. These analyses indicateda sedimentation coefficient of 3.6-3.8S, a Stokes radius of 25-28 A, and a calculated relative molecular weight of 42,000-

45,000 for the most abundant binding species. The other bindingspecies sedimented at 4-5S under high ionic strength conditionsand at 8-1 OS under low ionic strength conditions and had a

Stokes radius of approximately 60 A, a relative molecular weightof ~100,000, and an estimated concentration of 5-20 fmol/mgcytosolic protein. Binding of [3H]TCDD to this species was

displaceable by a 200-fold M excess of 2,3,7,8-tetrachlorodiben-

zofuran. Therefore, this species was tentatively identified as theTCDD receptor. The properties of the high-capacity binder of[3H]TCDD were found to be similar to the characteristics of a

protein previously purified from the rat ventral prostate, prostaticsecretory protein, which binds androgens as well as estramus-

tine, a nitrogen mustard derivative of estradiol. The binding ofestramustine to diluted prostatic cytosol was shown to be competitively inhibited by 2,3,7,8-tetrachlorodibenzofuran. Moreover,purified prostatic secretory protein bound [3H]TCDD, [3H]-benzo(a)pyrene, as well as [3H]-3-methylcholanthrene. It is sug

gested that binding to this protein is responsible for the high-

binding capacity of carcinogens in cytosol from the rat ventralprostate.

INTRODUCTION

As a chemical etiology of prostatic cancer recently has beensuggested (1, 2), several studies have addressed the issuewhether the prostatic gland possesses enzymes necessary forthe metabolic activation of chemical carcinogens. In the ratventral prostate of untreated rats, it has been shown that the

Received 4/24/85; revised 10/15/85; accepted 10/16/85.1Supported by grants from the Swedish Medical Research Council (Grant 03X-

6807), the Swedish Cancer Society, and the Swedish Board for Planning andCoordination of Research.

2To whom requests for reprints should be addressed.

cytochrome P-450-dependent microsomal enzyme activity AHH3

as well as Â¡mmunodetectable amounts of the major 0-naphtho-flavone-inducible isozyme of cytochrome P-450, cytochrome P-450c (the form mainly responsible for AHH-activity) are very low.Following treatment with /3-naphthoflavone or TCDD, an approximately 500-fold induction of cytochrome P-450c is detectable

by both immunochemical techniques and AHH activity determinations (3-7). Moreover, this induction in the prostate has been

shown to be accompanied by an increased capability to formmutagenic metabolites from the promutagens 2-aminofluoreneand B(a)P as determined by the Ames' Sa/mone//a/microsome

assay (6).A substantial amount of evidence indicates that the induction

of cytochrome P-450c in several tissues is mediated by binding

of the inducing agent to an intracellular, soluble receptor protein,which in a poorly understood process leads to an increasedaffinity of the ligand-receptor complex for target sites within the

cell nucleus (8). TCDD is one of the most potent agonists knowntoday for both the enzyme-induction response (9) and receptorbinding (10). Other agonists include such well-known carcinogens as 3-MC and B(a)P (10). The TCDD receptor has been

demonstrated in several extrahepatic tissues in both rats andmice (11,12). Surprisingly, no TCDD receptor has been reportedfor the rat ventral prostate, possibly indicating a nonreceptormediated induction process of cytochrome P-450c in this tissue.

However, a very high degree of nonspecific (nonreceptor) bindingof [3H]TCDD to an unidentified component in undiluted cytosol

from the rat prostate has been reported (11,12). It has also beenreported that [3H]B(a)P readily interacts with components in rat

prostatic cytosol (13). In this paper, we characterize the bindingof various radioligands ([3H]TCDD, [3H]-3-MC, and [3H]B(a)P) to

ventral prostatic cytosol and fractions thereof.

MATERIALS AND METHODS

Chemicals. [1,6-3H]TCDD (28 Ci/mmol) was a generous gift from Dr.A. Poland (Madison, Wl). [G-3H]3-MC (27 Ci/mmol) and [G-3H]B(a)P (25

Ci/mmol) were from the Radiochemical Centre, Amersham, England. [1-14C]-Glucose (8 Ci/mol) was from New England Nuclear Corp. (Boston,

MA). Unlabeled TCDF was kindly supplied by Dr. C. Rappe (UmeÃ ,Sweden). Estramustine and [2,4,6,7-3H]estramustine (107 Ci/mmol) were

generously provided by Leo Pharmaceuticals (Helsingborg, Sweden).Activated charcoal (Norit A), cytochrome c (equine heart), albumin (bovineserum fraction V), rabbit IgG, and catalase (bovine liver) were purchased

3The abbreviations used are: AHH, aryl hydrocarbon hydroxylase; cytochromeP-450C, the major form of cytochrome P-450 isolated from rat liver microsomes of0-naphthoflavone-(5,6-benzoflavone}-treated rats; TCDD, 2,3,7,8-tetrachlorodi-benzo-p-dioxin; 3-MC, 3-methylcholanthrene; B(a)P, benzo(a)pyrene; TCDF,2,3,7,8-tetrachlorodibenzofuran; estramustine, 1,3,5(10)-estratriene-3,17/i-diol-3-N-bis(2-chloroethyl)carbamate; DCC, dextran-coated charcoal; PSP, prostatic steroid binding protein; PAH, polycyclic aromatic hydrocarbon.

CANCER RESEARCH VOL. 46 FEBRUARY 1986

651

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CARCINOGEN-BINDING PROTEINS IN THE RAT PROSTATE

from Sigma Chemical Co. (St. Louis, MO). Sephacryl S-300, SephadexG-25 and G-100 fine, DEAE-Sepharose, heparin-Sepharose, blue dextran2000, and dextran T-70 were from Pharmacia Fine Chemicals (Uppsala,

Sweden). All other chemicals were analytical grade products from eitherSigma or Merck AG (Darmstadt, Federal Republic of Germany).

Animals. Male Sprague-Dawley rats weighing 200-300 g were used

throughout this study.Buffer. The following buffer was routinely used: 20 rnw potassium

phosphate-1 HIM EDTA-2 rriM 2-hydroxyethylmercaptan-10% (w/v) glyc-

erol, pH 7.2.Preparation of Cytosol. Rats were killed by cervical dislocation and

the two ventral prostate lobes were immediately excised and removedonto ice. All successive work was carried out at 0-4Â°C unless indicated

differently. The prostates were finely minced with a pair of scissors inapproximately 5-10 vol of buffer and thereafter homogenized in 2 vol ofbuffer in a teflon/glass Potter-Elvehjem homogenizer. The homogenate

was then centrifuged at 140,000 x gav for 45 min. The supernatant,hereafter termed cytosol, was removed and care was taken to avoid thefloating lipid layer.

Purification of PSP. The mincing procedure of the prostatic glandresulted in release of secretory fluid from the ductules and lumina (14)which subsequently together with the cytosol was used for purificationof PSP by anion-exchange and gel permeation chromatography as

described by Heyns et al. (15). Purified PSP was quickly frozen in aliquotsand stored at -70Â°C. In some cases, PSP was delipidated by precipi

tation in acetone (16,17) before assessment of ligand binding.In Vitro Labeling Conditions. For determination of TCDD receptor

binding, crude cytosol or various Chromatographie fractions of cytosolwere labeled with 3-5 nw [3H]TCDD at the indicated protein concentrations for 2-18 h at 0-4Â°C, usually in the absence or presence of a 200-

fold M excess of unlabeled TCDF. TCDF was used instead of unlabeledTCDD to determine specific binding in view of its greater solubility inwater and similar affinity for the receptor (10). Bound and free ligandwere usually separated by DCC adsorption [1.9% (w/v) charcoal and0.19% (w/v) dextran T-70, final concentrations] as previously described(18). In some cases, a hydroxylapatite-based assay for receptor binding

of TCDD (19) was used to separate free as well as specifically andnonspecifically bound [3H]TCDD. PSP was labeled with the indicatedconcentrations of radioligand at 37Â°C for 30 min, whereafter DCCadsorption was performed at 0-4Â°C (0.1-1% charcoal, final concentra

tions, as indicated). All receptor or PSP ligands were dissolved andadded to the incubations in dioxane, the concentration of which neverexceeded 1%(v/v).

Velocity Sedimentation Analysis. Labeled samples (200 n\) werelayered onto linear 5-20% (w/v) or 10-40% (w/v) sucrose gradients in

buffer (containing 0.4 M KCI in indicated cases), followed by centrifugationin an SW 60ri rotor in a Beckman L8-M centrifuge at 260,000-320,000x gavfor 14-18 h to a preset cumulative centrifugal effect (w^) of 1.7 x1012 rads2/s for TCDD receptor analysis on 10-40% (w/v) sucrosegradients (17) and 2.15 x 1012 rads2/s for PSP-binding analysis on 5-

20% (w/v) sucrose gradients. Fractions (s200 Ml/fraction) were collected

from the bottom of the centrifuge tubes by gravity flow. Cytochrome c(1.7S), bovine serum albumin (4.4S), -y-globulin (6.6S), and catalase(11.35) were 14C-labeled (20) and used as sedimentation markers. Sedi

mentation velocities of the receptor peaks were calculated according toMartin and Ames (21).

Gel Permeation Chromatography. Analytical gel permeation chromatography on 1- x 115-cm columns of Sephacryl S-300 was performedat a flow rate of 5 ml/cm2/h as previously described (18). The columns

were equilibrated in buffer containing 0.15 M NaCI and 0.02% (w/v)sodium azide. Sephadex G-100 was equilibrated in buffer containing

0.02% (w/v) sodium azide and packed into a siliconized column (1.5 x87 cm). The flow rate was 14 ml/cm2/h and the sample volumes were

approximately 1% of the total column volume. The columns were calibrated with the following standard proteins: thyroglobulin (86.1 A); ferritin

(61.5 A); catalase (51.3 A); bovine serum albumin (35.9 A); and cytc-chromec(17.9A).

Calculation of Hydrodynamic Properties. Relative molecularweights, and frictional ratios due to shape, f/f0, were calculated from theStokes radius, R5, and sedimentation coefficient as described by Siegeland Monty (22), assuming a partial specific volume of 0.725 cm3/g and

a solvation factor of 0.2 g/g of solute (18). Axial ratios, a/b, for hydro-dynamically equivalent prolate ellipsoids were derived from the valueslisted by Schachman (23). S and fls values for the protein standardswere obtained from the literature (24, 25).

Heparin-Sepharose Chromatography. Heparin-Sepharose was equil

ibrated in buffer and packed into a siliconized column (2.6 x 8 cm).Nonlabeled cytosol was applied to the column which was then washedwith 2-3 column vol of buffer, whereafter retained material was eluted

with 1 column vol of buffer containing 0.5 M NaCI at a flow rate of 4 ml/cm2/h.

Saturation Studies. Saturation studies were performed in siliconizedglass tubes at cytosolic protein concentrations of approximately 1 ^g/mlbuffer (26, 27) containing 0.1% (w/v) gelatin (26). Gelatin was includedin the incubation mixture in order to avoid adsorption of ligand or proteinto the wall of the incubation tube. Under these conditions, up to 10 nw[3H]TCDD and approximately 25 nw [3H]B(a)P and [3H]-3-MC, respec

tively, could be dissolved. Diluted cytosol or purified PSP were incubatedin 0.5-ml aliquots with varying concentrations of ligand at 37Â°C for 30

min. The tubes were removed onto ice, and unbound ligand was adsorbed by the addition of 0.5 ml of an ice-cold DCC suspension [1.5%(w/v) charcoal-0.05% (w/v) dextran T-70-0.1% (w/v) gelatin in buffer].

The incubations were agitated and left on ice for 10 min. The charcoalwas then pelleted by centrifugation at 1500 x g for 10 min, whereafterthe supernatant was assayed for radioactivity. To determine the totalradioactivity added, aliquots (generally, 20 ><l)of the incubations wereremoved shortly before the addition of the DCC. Binding data wereanalyzed by Scatchard plots (28) or double-reciprocal plots using linear

regression analysis to determine apparent equilibrium parameters ofbinding.

General Methods. Protein concentrations were determined using themethod of Lowry ef al. (29), with bovine serum albumin as the standard.Radioactivity was measured in a Scintillator 299 from Packard (DownersGrove, IL) in an LKB-Wallac Rackbeta II scintillation spectrometer (Stock

holm, Sweden) with an average counting efficiency of 40%.Safety Precautions. Since TCDD and TCDF are extremely toxic

compounds (8), their use necessitated special handling and disposalprocedures as outlined by Poland ef al. (10).

RESULTS

By means of gel permeation chromatography of [3H]TCDD-

labeled crude rat ventral prostatic cytosol on Sephacryl S-300 itwas possible to distinguish between two discrete [3H]TCDD-

binding species which both were included into the gel (Fig. 1).The first peak of radioactivity with an f?s of approximately 60 Awas nearly completely abolished by labeling of the crude cytosolin the presence of a 200-fold M excess of TCDF. The 60-Ã•

binding entity was, however, partly obscured by high levels of amajor [3H]TCDD-binding species eluting in the 25- to 28-Ã• region

of the column. Under these experimental conditions, binding of[3H]TCDD to this entity was nondisplaceable (nonspecific) anddisturbed the further characterization of the 60-Ã‚ [3H]TCDD-

binding species. It was therefore investigated whether heparin-Sepharose chromatography of nonlabeled cytosol could removeany interfering nonspecific binding. It has previously been established that the TCDD receptor readily interacts with heparin-Sepharose both in the presence or absence of ligand (18). It waspossible to label the material eluted from heparin-Sepharose with


652




-10 -

20 40 60 80 100

ELUTION VOLUME Imi!

Fig. 1. Gel permeation chromatography of rat prostatic cytosol labeled with[3H]TCDD.Prostatic cytosol (=10 mg protein/ml) was incubated with 5 nM [3H]TCDD for 2 h at 0-4Â°Cin the absence (O) or presence (â€¢)of a 200-fold Mexcessof unlabeled TCDF. Bound and free ligand were separated by adsorption toprepelletedDCC [final concentration of charcoal, 1.9% (w/v)]. A sample volume of1 ml was then chromatographed as described under "Materials and Methods."

Fractions (2.0 ml) were collected and assayed for radioactivity. The columns werecalibrated with blue dextran 2000 (V0),[uC]glucose (V,)and the following standardproteins: ?, thyroglobulin (86.1 A); 2, ferritin (61.5 Ã€);_3,catalase(51.3 Ã€);4, bovineserum albumin (35.9 A); and 5, cytochrome c (17.9 A).

2.0

>â€¢1.0

0.5

3 2 1

I I I

SB*

3 2 1

10 20

FRACTION

20top

Fig. 2. Velocity sedimentationanalysisof [3H]TCDD-labeledheparin-Sepharoseeluates. Nonlabeled prostatic cytosol (5 ml; =50 mg protein) was applied to aheparin-Sepharose column and then washed with 2-3 column vol of buffer.Retained material was eluted with 1 column vol of buffer containing 0.5 M NaCIand desalted on Sephadex PD-10 columns. The eluates were incubated with 3 nu[3H]TCDDfor 2 h at 0-4Â°C,in the absence (O) or presence (â€¢)of a 200-fold Mexcess of unlabeled TCDF. Bound and free ligand were separated by DCCadsorption [final concentration of charcoal, 1.9% (w/v)]. Aliquots (200 >il)were thenlayered onto 10-40% (w/v) sucrose gradients prepared in buffer (A) or in buffercontaining 0.4 M KCI (B). The gradients were centrifugea and fractions werecollected as described under "Materials and Methods." Sedimentation markers

were: 1, cytochrome c (1.7S); 2, bovine serum albumin (4.4S); 3, IgG (6.6S); and4, catalase (11.3S).

[3H]TCDD and detect specifically bound radioactivity by Sephac-

ryl S-300 gel permeation chromatography (data not shown) andsucrose gradient analysis (Fig. 2). The velocity sedimentation ofthe specific [3H]TCDD-binding entity varied as a function of the

ionic strength. At low ionic strength, it sedimented as an 8-1 OS

entity (Fig. 24), whereas at high ionic strength (0.4 M KCI), itsedimented as a 4-5S entity (Fig. 2B). In the heparin-Sepharose

eluates (Fig. 2) as well as in crude cytosol (data not shown),nonspecific [3H]TCDD binding was detectable as a slower sedi-menting entity of 3.6-3.8S under both high- and low-salt conditions. The fls value of the specific [3H]TCDD-binding entity in

prostatic cytosol and its sedimentation coefficient of 4-5S indi

cates a highly assymetrical molecule with a molecular weight ofapproximately 100,000 an f/f0 of 1.7-1.8, and an a/b of 12-15.

All of these hydrodynamic properties are indistinguishable fromthe physicochemical characteristics of the TCDD receptor present in other tissues of the rat (18, 30-32). The concentrations ofthe specific 60-Ã‚ [3H]TCDD-binding entity varied between 5-20

fmol/mg of cytosolic protein, as estimated by high-resolution gelpermeation chromatography on Sephacryl S-300. These observations were confirmed by analysis of [3H]TCDD-labeled ventral

prostatic cytosols by a hydroxylapatite assay (19) which indicated TCDD receptor concentrations of approximately 10-12

fmol/mg protein. In this assay, 175 HIM phosphate was sufficientto extract all the specifically bound [3H]TCDD which initially had

been immobilized on hydroxylapatite (data not shown). This is inagreement with the behavior of the TCDD receptor on hydroxylapatite in other tissues, where the TCDD receptor Ã©lÃ»tesfromhydroxylapatite at 150-175 rriM phosphate (18,19). Quantitationof specifically bound [3H]TCDD in labeled heparin-Sepharoseeluates yielded concentrations of the specific [3H]TCDD binding

species of -15-20 fmol/mg protein. However, this value might

be artificially low due to, e.g., low recovery of the binding entityduring the heparin-Sepharose chromatography or difficulties in

labeling partially purified TCDD receptor preparations (33).The hydrodynamic properties of the major [3H]TCDD-binding

entity in undiluted cytosol from the rat ventral prostate (fls ~25-28 A; 3.6-3.8S; cf. above) indicate an apparent molecular weightof 40,000-45,000, an f/f0 of 1.0-1.1, and an a/b of 2-5 which is

characteristic of a nearly symmetrical molecule. It has also beenshown that [3H]TCDD interacts with a binding species in rat

ventral prostatic cytosol which focuses at a pH of about 4.7-5

during isoelectric focusing in polyacrylamide gels and which isdistinct from or obscures the TCDD receptor (11). All of thesedata (summarized in Table 1) closely resemble the physicochem-

Table 1Comparisonof physicochemical properties of the major pHJTCDD-binding

species in rat prostatic cytosol and PSPPhysicochemicalcharacteristics of the major rat prostatic [3H)TCDD binding

species were experimentally determined or calculated as described in "Materialsand Methods." Properties of PSP were from the literature or in given cases

calculated by the equations used in the text.

ParameterS(s-")

M, .fl.(A)fifaAxial ratioIsoelectricpointMajor

[3H]TCDD-bindingspecies3.6-3.8

40,000-45,00025-281.1-1.22-54.7-5.0"PSP3.7"

38,000i>-46,000c29"1.3*5-8"4.6-4.9'

" Heyns and De Moor (17).6 Calculatedsum of the molecularweight of the different PSPsubunits (39).c Forsgrener al. (37)." Calculatedvaluesfrom the sedimentationcoefficient (17)and molecularweight

(37) (cf. "Materials and Methods").eCartstedt-Duke(11).' Heynsef al. (36).


653




Â¡calcharacteristics of a major secretory protein in the rat ventralprostate, often referred to as PSP (17, 34). In view of these datawe examined the binding of various TCDD receptor ligands tocrude cytosol as well as to purified PSP.

When crude prostatic cytosol or purified PSP was labeled withsubsaturating concentrations of [3H]TCDD at 37Â°C, a rapid

association of ligand could be observed, as determined by DCCadsorption. After 30 min, maximal association was reached andrepresented approximately 85% of the total radioactivity. Thereafter, charcoal-resistent binding slowly declined, so that after 24h approximately 10% of the maximal binding remained. At 0Â°C,

the association rate was slow; first after 24 h, a plateau wasreached both in crude cytosol and incubations using pure preparations of PSP. However, in both cases this binding representedonly approximately 30-40% of the maximal binding observed at37Â°C(data not shown). Analysis of the purified PSP preparations

on 5-20% (w/v) sucrose gradients revealed a single symmetricalpeak of radioactivity in the same region of the gradients (3.5-4S) (Fig. 3A) as the major [3H]TCDD-binding entity in crude

cytosol (cf. above). The radioactivity recovered under this peakaccounted for approximately 78% of the total radioactivity. Itwas also possible to label purified PSP with [3H]-3-MC or [3H]

B(a)P, as determined by velocity sedimentation (Fig. 36). In thecase of [3H]-3-MC and [3H]B(a)P, 65 and 46% of the total

radioactivity were recovered, respectively, on the sucrose gradients.

Saturation of binding of [3H]TCDD and [3H]estramustine to the

major binding species was obtained only when cytosol wasdiluted to a protein concentration of approximately 1 Â¿ig/mlinthe presence of 0.1% (w/v) gelatin. This binding was furtherevaluated by Scatchard analysis (28). Linear Scatchard plots (ra 0.9) were obtained for the binding of both compounds. This isin agreement with a single class of binding sites with uniformaffinity for the tested ligand. Typical plots are shown in Fig. 4.Both [3H]TCDD and [3H]estramustine bound to the same maximal number of sites (~1 nmol/mg cytosolic protein; s5% of the

10 20top

20

FRACTION

Fig. 3. Velocity sedimentation analysis of purified PSP labeled with [3H]TCDD(A), or [3H]-3-MC (O) and [3H]B(a)P (â€¢)(B). Purified PSP (a1 mg protein/ml) wasincubated with 5 nM [3H]TCDD, 5 nw [3H]-3-MC, or 5 nw [3H]B(a)P at 37Â°Cfor 30

min. Bound and free ligand were separated by DCC adsorption [final concentrationof charcoal, 1% (w/v)]. Aliquots (200 pi) were then layered onto 5-20% (w/v)sucrose gradients prepared in buffer. The gradients were centrifuged and fractionswere collected as described under "Materials and Methods". Sedimentation mark

ers were: 1, myogtobin (2.0S); 2, ovalbumin (3.5S); and 3, bovine serum albumin(4.4S).

<U

0.2

0.5

BOUND

1.0

I nM 1Fig. 4. Representative Scatchard plots comparing binding of [3H]estramustine

and [3H]TCDD to rat ventral prostatic cytosol. Cytosol (s1.3 pg protein/ml) wasincubated in the presence of 1% (w/v) gelatin at 37Â°Cfor 30 min with increasingconcentrations of [3H]estramustine, 0-10 nu (O) or [3H]TCDD, 0-5 nM (D)- Theincubations were terminated by the addition of 0.5 ml of an ice-cold DCC suspensionyielding a final concentration of charcoal of 1% (w/v). Subsequent to DCC adsorption (see "Materials and Methods"), free ligand was calculated by subtracting bound

ligand from the total concentration of ligand present in the incubation. From thesedata, Scatchard plots (25) were constructed by least squares linear regressionanalysis.

4.0

3.0o *-Z 2-

2.0

0.5 1.0{UNBOUND)'1

1.5l n M'

Fig. 5. Competitive binding of TCDF to [3H]estramustine-binding sites in prostatic cytosol. Cytosol (=1.3 tig protein/ml buffer containing 0.1% gelatin) wasincubated at 37Â°Cfor 30 min with 0.5-1.0 nM [3H]estramustin in the absence (O)

or presence of 5 (â€¢)or 10 (D) nw unlabeled TCDF. Specific binding was determinedby DCC adsorption [final concentration of charcoal. 1% (w/v)] as described under"Materials and Methods," and the obtained data were plotted according to Line-

weaver and Burk (35) using linear regression analysis. Points, mean of duplicatedeterminations.

total cytosolic protein content), suggesting binding of the twoligands to the same site in cytosol. The apparent Ka in thisparticular experiment was 4.1 nM for [3H]estramustine and 1.9nw for [3H]TCDD.

To further investigate if halogenated PAH interacted with thesame class of binding sites in diluted prostatic cytosol as did[3H]estramustine, binding of various concentrations of [3H]estra-

mustine was determined in the absence or presence of 5 or 10nM TCOF, respectively. TCDF was chosen as a structural analogue of TCDD because of its more pronounced solubility inwater (10). The binding data obtained were analyzed accordingto Lineweaver and Burk (35) and were found to be compatiblewith a competitive inhibition of [3H]estramustine binding by TCDF(Fig. 5). An apparent Ka of 4.6 nM could be calculated for [3H]estramustine from the double-reciprocal plot in Fig. 5, which is

in good agreement with the value determined by Scatchard plots(cf. above). Moreover, the number of binding sites determinedby double-reciprocal plot analysis in this experiment was approx

imately 0.7 nmol/mg cytosolic protein.


654




Titration of diluted prostatic cytosol with [3H]-3-MC or [3H]-

B(a)P revealed nonlinear, upward concave curves when apparentequilibrium binding data were analyzed according to Scatchard(28) (Fig. 6) indicating at least two classes of binding sites.Interestingly, in addition to low-affinity binding, two straight linescould be extrapolated from the concave graphs (r s 0.90 and0.92, respectively) indicating the presence of a high-affinity binding entity with a Ka of approximately 2 nw for [3H]-3-MC and [3H]

B(a)P, respectively. In addition, these lines seem to have anintercept with the abscissa in the Scatchard plots similar to thevalues obtained for [3H]TCDD and [3H]estramustine (cf. Fig. 4).

DISCUSSION

In the present investigation, equilibrium binding studies indicate the presence of a major, saturable, high-affinity binding sitefor [3H]TCDD in diluted cytosol from the rat ventral prostate at

a concentration of about 1 nmol/mg of total cytosolic protein. Incontrast to this observation, hydrodynamic characterization bygel permeation chromatography and sucrose gradient sedimentation analysis indicates that [3H]TCDD binding to rat prostatic

cytosol is composed of at least two discrete binding species.The major fraction of bound [3H]TCDD was associated with a

symmetrical molecule of 3.6-3.8S, an Rs of 25-28 A, and amolecular weight of approximately 40,000-45,000. These phys-

icochemical characteristics agree well with the properties of PSP,a protein purified by several laboratories from the rat ventralprostate. (36-38). PSP is a tetrameric glycoprotein with anisoelectric point of about 4.6-4.9 (36) consisting of two subunits,one containing the two different polypeptides termed CI (M, ~10,200) and C3 (M, ~ 8,600) and the other containing C2 (M, ~

10,500) and C3 (34, 39). The carbohydrate content of PSP hasbeen estimated to be 3.2% (36). The most prominent functionalcharacteristic of PSP appears to be steroid binding (17, 36).Androgens induce PSP synthesis (40, 41) and bind to PSP withlow affinity (Ka ~1 pM) (17, 34). Moreover, it is known that

estradici mustard derivatives which are used in treating prostaticcarcinoma (42) bind to PSP with high affinity (Ka~10-30 nw) (26,

27, 37). The biological function of PSP is presently not known.

CL O.It -

0.2 -

0.5 1.0 1.5

BOUND I n M I

Fig.6. Scatchardanalysisof the bindingof [3H]B(a)Pand [3H]-3-MCto prostaticcytosol. Cytosol (si .3 Â»igprotein/ml buffer containing0.1% gelatin)was incubatedat 37Â°Cfor 30 min with increasingconcentrations (0-20 nM)of [3H]B(a)P(D) and|3H|-3-MC (O) before binding was assayed as described under "Materials andMethods" and in the legend to Fig. 4. Points, mean of duplicate determinations.

The broken lines were extrapolated from the graphs by linear regression analysis(r ~ 0.90-0.92) to obtain tentative Kasfor a possible high-affinitycomponent.

Thus far, it has been shown that the C2 polypeptide (cf. above)can bind steroids (36, 38) and that the CI and C2 chains exhibitamino acid sequence homology with each other (34) and withrabbit uteroglobin (39), whereas the C3 chain exhibits amino acidhomology with human a2-macroglobulin (43). In the presentstudy, [3H]TCDD bound to a saturable binding species in crude

cytosol with high affinity (Kâ€žs 2 nM). Several lines of evidencesupport the contention that this binding species in fact representsPSP: (a) both [3H]estramustine and [3H]TCDD interacted with

the same maximal number of binding sites in crude cytosol asdetermined by Scatchard plot analysis; (D)double-reciprocal plot

analysis according to Lineweaver and Burk (35) showed thatTCDF, a structural analogue of TCDD, competitively inhibitedestramustine binding to crude cytosol; (c) the physicochemicalproperties of the major [3H]TCDD-binding entity in crude cytosolclosely resemble those reported for PSP (cf. above); and (d) [3H]TCDD as well as [3H]B(a)P and [3H]-3-MC readily interacted with

purified PSP as assayed by velocity sedimentation analysis.Extrapolation of equilibrium binding data calculated according

to Scatchard (28) indicated that both [3H]B(a)P and [3H]-3-MC

bound to a similar saturable binding species in diluted cytosol as[3H]TCDD and possibly with a similar affinity (Ka s 2 nM). It haspreviously been shown that [3H]B(a)P and [3H]-3-MC interact

with a 4S, high-capacity, high-affinity carcinogen-binding proteinin rat liver cytosol with very similar affinities (Ka = 2.5 and 2.8nM, respectively) (44,45). A similar protein binding [3H]B(a)P with

a Ka of 1.8 nM has recently been described in a mouse embryocell line (46).

The mechanism of induction of cytochrome P-450c in the rat

ventral prostate is intriguing. There exists an increasing amountof data on the induction of AH H activity and cytochrome P-450cin the rat ventral prostate (3-7). However, no TCDD receptor

has been observed in this tissue in the rat (11,12), whereas thereceptor has been detected in mouse prostatic cytosol4 (12)

suggesting a nonclassical nonreceptor mediated induction process of cytochrome P-450c in the rat prostate. Interestingly,analytical gel permeation chromatography of [3H]TCDD-labeledcrude cytosol also indicated binding of [3H]TCDD to a discrete

component in cytosol with an fls of ~60 A. Moreover, prechro-matography of unlabeled cytosol on heparin-Sepharose revealedthe presence of a specific [3H]TCDD binding species which

sedimented at 8-1 OS under low-ionic strength conditions and4-5S under high-ionic strength conditions and which was hardly

distinguishable in crude cytosol. This salt dependent sedimentation shift is characteristic of the TCDD receptor Â¡nseveraltissues (18,30) and of steroid hormone receptors in general (47).Furthermore, the physicochemical characteristics of this bindingspecies, i.e., sedimentation coefficients, fls, and molecular weightare identical to those observed in rat liver cytosol for the TCDDreceptor (18, 33). In view of these data, we believe that thisparticular binding species represents the TCDD receptor andthat its detection in crude cytosol is severely hampered by thehigh abundance of high-affinity [3H]TCDD-binding to PSP. Inspite of the abundance of the major [3H]TCDD-binding species,

analysis of crude cytosol by gel permeation chromatographyindicates the presence of the receptor. Several explanations areconceivable for this observation, e.g. (a) a subsaturating concentration of [3H]TCDD with regard to binding to the major binding

4P. Soderkvist, L. Poellinger,and J-Ã•.Gustafsson, unpublishedresults.


655




species and (b) differences in affinities of the TCDD receptor andthe major binding species, respectively. For instance, the rathepatic TCDD receptor has a Ka for [3H]TCDD of 0.2-0.8 nM

(19). Moreover, no information is available at the present on thedissociation rates of [3H]TCDD from either the major binding

species or the TCDD receptor. Gel permeation chromatographyanalysis on Sephacryl S-300 was carried out during approxi

mately 24 h. Therefore, the loss of bound ligand due to dissociation and association to the gel matrix might be considerable.

The pronounced abundance of PSP in prostatic epithelial cells(S20% of the total protein content in cytosol) and in seminal fluid(17) confers a potentially high binding capacity for chemicalcarcinogens like B(a)P and 3-MC as well as for toxic substances

such as chlorinated dibenzodioxins and dibenzofurans and mightcause the accumulation of these and possibly related substancesin the prostate. High-capacity binding of the chlorinated hydrocarbon insecticides dieldrin and o,p'-dichlorodiphenyltrichloro-

ethane to a 3.5S androgen-binding protein in cytosol from the

rat ventral prostate has previously been described (48). Theproperties of this protein are similar to those of PSP (cf. Table1). In vivo studies have shown that o,p'-dichlorodiphenyltri-

chloroethane accumulates in the mouse prostate (49). A 3-4Scarcinogen- and TCDD-binding protein has been observed in

mouse prostatic cytosol, albeit at lower concentrations as compared to in rat prostatic cytosol4 (12). Furthermore, in vivo uptake

and secretion of 3-MC by the rat and dog prostates has been

demonstrated (50). Thus, it is conceivable that PSP is of relevance for the tissue-specific accumulation and secretion of struc

turally related PAH.It is interesting that a secretory protein exhibiting amino acid

homology with PSP, uteroglobin (39) (cf. above), also bindssteroids with a similarly low affinity as compared to PSP (Ka s 1nM) and polychlorinated biphenyl congeners such as 4,4'-bis(methylsulfonyl)-2,2',5,5'-tetrachlorobiphenyl with high affin

ity (Ka = 2.5-15 nM) (51). The physiological roles of either PSPor uteroglobin or the toxicological implications of the PAH-protein

interaction are presently not understood. Furthermore, it is notknown if high-capacity binding of PAH by e.g. PSP is of importance for the mechanism of induction of cytochrome P-450c.However, it has been suggested that carcinogen-binding mole

cules in rat liver may participate in the microsomal metabolismof PAH as intracellular transport proteins (52, 53). Unfortunately,these molecules have not yet been identified or purified. It isconceivable that PSP might be functionally similar to such amolecule in rat liver. Therefore, the rat ventral prostate mightpresent an attractive model for further studies of the mechanismof induction of cytochrome P-450c and the microsomal metab

olism of PAH.

ACKNOWLEDGMENTS

We thank Dr. A. Poland (University of Wisconsin, Madison, Wl) for providing[3H]TCDD, Dr. C. Rappe (Univerity of UmeÃ ,Sweden) for unlabeled TCDF and Dr.B. Forsgren (AB Leo Research Laboratories, Helsingborg, Sweden) for [3H]estra-

mustine. Dr. J. Lund (Karolinska Institute, Stockholm, Sweden) is gratefully acknowledged for fruitful discussions throughout the study.

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1986;46:651-657. Cancer Res Peter Söderkvist, Lorenz Poellinger and Jan-Åke Gustafsson

-dioxinp2,3,7,8-Tetrachlorodibenzo-)pyrene, 3-Methylcholanthrene, anda

Specific and Nonspecific High-Affinity Binding Sites for Benzo(Carcinogen-binding Proteins in the Rat Ventral Prostate:

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