In vivo binding of diethylstilbestrol to nuclear proteins of kidneys of Syrian hamsters

CANCER LETTERS Cancer Letters 90 (1995) 2 15-224

ELSEVIER

In vivo binding of diethylstilbestrol to nuclear proteins of kidneys of Syrian hamsters Deodutta Roy*, Deena Nath Pathak’, Murali Palangat Environmental

Toxicology

Program,

Department Birmingham,

of Environmental Alabama 35294-0008.

Health USA

Sciences,

University

of Alabama,

Received 17 December 1994;revision received 3 January 1995; accepted 19 January 1995

Abstract

Wedemonstrateherethat stilbeneestrogen(diethylstilbestrol)is convertedto nuclearprotein bindingmetabolite(s) both in vitro and in vivo. In vitro reactionof DES with nucleifrom hamsterliver or kidney in the presenceof cumene hydroperoxideor NADPH revealedbinding of [3H]DES in nuclearproteins(histones;nonhistonesprecipitableby 2%TCA, NH2; nonhistones solublein 2%TCA, NH30). The bindingwassignificantlyinhibited by cytochromesP450 inhibitors. In an in vitro system[‘HIDES quinone, one of the metabolitesof DES, was able to bind to pure nonhistoneproteinsRNA polymeraseand DNA polymerase.The binding of [‘HIDES quinoneto nonhistonesRNA polymeraseand DNA polymerasewasinhibited by low molecularweightthiols, i.e. glutathioneand cysteine,or thiol modifiers,suchasn-ethylmaleimide,dithionitrobenzoic acid and hydroxymercuric benzoate.DES and DES metabolitesinhibited transcriptionalactivity. In vivo [3H]DES wasableto bind to nuclearproteinsof hamsterliver, kidneys and testes.The level of in vivo [3H]DES binding to all three types of nuclearproteins(histones,NH2, NH30) in the kidney (target organ)wastwo or morefold higher than that observedin the liver or testis(nontargetorgans).Four nuclearNH30 proteins(mol wts.: 56, 37, 33 and 28 kDa) were irreversibly bound to [3H]DES in vivo. The in vivo bindingof [3H]DES to transcriptionally active chromatinNH30 proteinsalsowasobserved.The data reportedhere establishthat DESwasableto bind to liver or kidney nuclearproteinsin vitro, whichwascatalyzedby nuclearenzymes whenfortified with an appropriatecofactor. DES quinonemay be one of the protein binding metabolites.DES and DES metabolitesinhibited transcriptionalactivity. The level of in vivo binding of [3H] DES to nuclearproteinsof kidney (target organ) was double in comparisonwith that observedin liver or testis(nontarget organs).In vivo modificationsin the chromatin proteinsmay be a factor in the developmentof DES-inducedrenalcarcinogenesis is not clear. Keywords:Diethylstilbestrol;Liver; Kidney; Nuclear proteins; Syrian hamsters

Abbreviations:

DES, diethylstilbestrol; NH2, nonhistones

precipitable by 2%TCA; NH30,nonhistones solublein 30% TCA; ANIT, cr-naphthylisothiocyanate; PMSF, phenylmethylsulfonyllluoride; OA, n-octylamine. Elsevier Science Ireland Ltd. SSDI

0304-3835(95)03706-3

l

Corresponding author, Tel.: +I 205 9346081;

Fax:+I 2059756341. t Present address: Brain Tumor Research Center, IJniversity of California, San Francisco, CA, USA.

1. Introduction Diethylstilbestrol (DES), a synthetic estrogen. is B carcinogen. Chronic administration of DES pro. duces 80.- 100% tumors in the kidneys of SyriaIt hamsters [I]. The mechanism of DES-induced car,cinogenesis is not clear. DES has been shown to covalently bind to DNA in vivo [2.3]. The level of DES-DNA adducts was lower in the kidney (target organ) compared to that of liver (non target) and the pattern of repair of adducts was similar in both target and nontarget organs [3,4]. Tumors appear specifically in the kidneys, not in the liver, of intact male hamsters by DES treatment ]l.3,5]. The in VIVOlevel of DNA adducts and repair did not provide any information to explain the specificity of tumor development. It appears that in addition to DNA modifications there are other factor(s) which may be involved in the DES-induced carcincl.. genesis. Recently, we have shown that redox cycling of DES is catalyzed by nuclear enzymes 161. tn an in vitro system, DES, in the presence of rat liver nuclei and cofactor, covalently binds to transcriptionally active chromatin histones and nonhistones [7,8]. It is not known whether DES binds to nuclear proteins of the target organ of cancer in viva and if it plays a role in the induction of DES-induced carcinogenesis. Covalent modification of the nuclear nonhistone proteins involved in the regulation of gene expression or transcription can play an important role in the conversion of normal cells into transformed cells [9]. Covalent modifications of stable nuclear nonhistone DNA binding proteins and/or transcription regulating proteins by chemical carcinogens could have a significant impact on several aspects of gene functions (viz., gene replication. transcription or repair). Therefore, in the present study we have examined the potential of DES to bind covalently to nonhistone and histone nuclear proteins both in vitro and in viva. Our results show that DES binds to both histones and nonhisiones. Covalent attack of DES metabolites Ronthe target organ nuclear proteins was higher !han that of nontarget organ 2. Materials and methods : !. C.“hemicdr

Phenylmethylsulfonylfluoride

(PMSF).

diethyl-

stilbestrol (DES ). cr-naphthylisothiocyanate (ANIT). n-octylamine (OA), SKF-525A, reduced glutathione (GSH). cysteine, n-ethylmaleimide, /)-hydroxymercuric benzoate, dithionitrobcnzoic acid, histone, and micrococcal nuclease were purchased from Sigma Chemical Co.. St. Louis? MO. Pure nonhistone proteins, RNA polymerase (E. co/i) and DNA polymerase I (E. co/i). were purchased from Boehringer Mannheim. [“HIDES (specific activity = 74 Ciimmol) was purchased from Amersham Corporation. Arlington Heights, II 2.2. Preparation

of’ nuclfi

Syrian hamsters (male, X weeks old from Charles River Inc.) received an i.p. injection of 50 mg/kg DES (containing 25 PCi) [‘HIDES). After 4 h untreated and DES treated animals were killed and liver. kidney and testis were excised. The nuclei were isolated according to the procedure of Blobel and Potter [IO]. In brief, tissue was homogenized in 2 vols. of 0.25 M sucrose solution containing 1.0 mM PMSF. The homogenate was filtered through cheesecloth and centrifuged for IO min at 1000 x s. The pellet obtained was suspended in 0.25 M sucrose and 4 vols. of 2.3 M sucrose was underlaid. The nuclei were sedimented by centrifugation at 100 000 x g for 60 min. The nuclei were then washed with 0.25 M sucrose containing 0,2 mM PMSF and the purity of nuclei was checked by phase contrast microscope after staining with eosin and hemotoxylin and also by electron microscopy [IO]. The nuclear pellets were fixed in situ and then fixed with 0~0~ buffered with phosphate buffer, dehydrated and embedded in a mixture of Araldite and Epon. Sections were stained with lead citrate. The serial sections of nuclear pellets were viewed under an electron microscope. Glucose Gphosphatase was assayed as described by Niranjan et al. [I I]. The amount of DNA and RNA was assessed by the method described previously [6]. The preparation of nuclei is critical for this study and therefore the purity of the nuclear preparations was assessed both biochemically and morphologically. The activity of glucose 6phosphatase in the microsomes (a positive control) was about 650 pmol/mg protein, which is similar to the previous report [I I]. However, we failed to

D. Roy et al. /Cancer

detect the activity of glucose 6-phosphatase (an enzymatic marker of endoplasmic reticulum) in the nuclei. The amount of DNA and RNA was 92-95% and 3-4% of total values, respectively. The treatment of nuclei with Triton X-100 (2%), effective in removing the outer nuclear membrane, failed to remove DNA or RNA from nuclei. The electron microscopic examination of nuclei suggested that the nuclei were highly pure and cellular contamination was not visible (Fig. 1). All values are in good agreement with the previous observations reported by us [6-81 and others [lo], and suggest that nuclei were highly pure. 2.3. Nuclear activation system The reaction consisted of nuclei (10 mg protein equivalent purified untreated hamster liver or kidney nuclei), 0.2 mM PMSF, 1 mM NADPH or 1.5 mM cumene hydroperoxide, and O-200 PM of DES (containing 0.5 &i [3H]DES) in a final volume of 1 ml 10 mM phosphate buffer, pH 7.5 [7,8]. Reactions were carried out for 30 min at 37’C. During each reaction, a control was run in parallel in the absence of cofactor or in the presence of boiled nuclei. Some of the reactions were carried out in the presence of cytochromes P450 inhibitors: 1 mM ANIT, OA or 0.5 mM SKF-525A. 2.4. Binding of f3HjDES to nuclear proteins After the binding reaction, the mixture was centrifuged at 1000 x g for 10 min. Histones were isolated by the method of Hnilica [12] as described previously [8]. The nuclear pellet insoluble in 0.24 N HCl was dissolved in 3 ml phosphate buffer, pH

Fig. I. An electron micrograph of nuclei ( x 21 000). The nuclei were isolated and prepared for electron microscopy as described previously [IO].

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90 (1995J

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7.5, containing 0.2% Triton and after 1 h, proteins were precipitated with 2% TCA [13]. This precipitate was designated as NH (i.e. nonhistones precipitable by 2% TCA). The 2% TCA soluble proteins were precipitated with 30% TCA and the pellet was designated as NH30 [13]. Both histones and NH were washed with methanol (x 5) followed by methanol/ether (4: 1, x 4-6) to remove free 13H]DES and its metabolites. The NH30 pellet was washed with ether ( x 4). Washing with organic solvents was continued until the radioactivity was not detected in the supernatant [14]. Some of the samples were further extracted with ethanol/ether (3: 1). Some samples were redissolved in 1.O N NaOH, reprecipitated with trichloroacetic acid and extracted with 80% methanol or 25% ether. All of these procedures failed to remove the bound radioactivity. 2.5. Gel electrophoresis The nuclear proteins were electrophoresed in slab gels containing 15% polyacrylamide by the method of Laemmli [ 151. Following electrophoresis, gels were stained with coomassie blue and were then sliced into l-mm slices. Each slice was treated with 0.5 ml of 30% hydrogen peroxide at 50°C for 20 h and radioactivity measured by LS counting. 2.6. Reaction of pure RNA or DNA polymerase with r3H]DES quinone, one of the metabolites of DES [3H]DES quinone was synthesized as described previously [6]. Each of the pure nonhistone protein (50 pg) was reacted with 100 PM DES quinone (8000 counts/min/pmol) in 1 ml of 10 mM phosphate buffer, pH 7.5 at room temperature for 1 h [8]. Free DES quinone and rearrangement products were extracted with organic solvents as described above. The radioactivity incorporated in protein was counted. 2.7. Binding of L3H’jDES to NH30 proteins in transcriptionally active chromatin After the binding reactions were done as described above, nuclei were digested with 50 units of micrococcal nuclease for 10 min, and the transcriptionally active chromatin was isolated according to the procedure reported previously [ 161. The NH30 was obtained by precipitating with 30%

TCA

from the supernatant devoid of histones and NH proteins, washed

tion counting. The binding is expressed as pmol/ mg protein/30 min.

2.8. Assay of’ transcription uctivii.r, Transcriptional activity was measured by the method of Yu et al. [ 171. The reaction mixture consisted of nuclei (100-125 gg DNA), SO mM Tris-HCI, pH 7.9, 2 mM MnCI,, 300 mM (NH&SQ+ 200 PM of NTPs and I PCi [LX‘*P]GTP. cr-Amanitin was added at a final concentration of 20 &ml or 200 @ml to inhibit RNA pol II and RNA pol III: respectively. After the reaction, an aliquot was plated on a Whatman 3-mM disc and immediately immersed in ice cold TCA (5%). TCA precipitahle radioactivity was measured after extensively washing the filters in cold TCA ( x4) followed by ether/ethanol i!: I) and ether In vitro effect of DES or DES plus NADPH on the transcriptional activity was studied by incubating purified nuclei with DES or DES plus NADPH for IO or 30 min at 37°C: followed by transcriptional assay

3. Resutts

3. I, In vitro binding Incubation of [‘HIDES with liver nuclei in the presence of cofactor cumene hydroperoxide or NADPH produced 55 or 20 times. respectively, higher binding to nuclear proteins, histones. NH2 (2% TCA precipitable nonhistones) and NH30 (2% TCA soluble nohistones), than that observed in the absence of cofactor or boiled nuclei (12-19 pmol/mg protein!30 min) (Table I ). The binding of [‘HIDES to nuclear proteins in the absence of cofactor did not change with increasing time of incubation (O-45 min) or increase in the DES concentrations (O-100 FM) (data not shown). Therefore, binding of [3H]DES to nuclear proteins in the absence of cofactor was considered as background. All the values of binding reported herein represent those obtained after subtraction of nonspecitic background binding (i.e. binding in the absence of a cofactor or boiled nuclei). Incubation of hepatic nuclei with [“HIDES in the presence of cumene hydroperoxide resulted in

29. Measurement of radioactivit?, Radioactivity was determined by liquid scintilla-

!n vitro binding !:I the presence

of 13H]DES of hamster

to hiatones. liver nuclear

I‘ondirron?.

nonhistones activation

Binding

precipitahlc system

of DES

IO various

by .?“+. f(‘r\

nuclear

!NH?)

proteins

and

nonhistoric<

Ipmolimg

protein!30

YH?

Histones

The reaction DES

were carried t)r 0.5 mM i\o:ated nuclear

mixture (4000

consisted

countdminlpmol

out in the presence SKF-52SA). Reaction

[8,13], proteins

washed with is expressed

of IO mg purified [‘HIDES) of cytochromes mixtures were

hamster

m, the final

liver volume

P450 inhibitor incubated for

organic solvent as described as pmolimg protein 130 min

nuclei.

‘WA

(NH.30)

min)

._.. - .--.-.-.-..-----.-~-

I 15.0 * IO.6

100 pM

rn 2’L

NH30

_- . .....-. -.._.-----...

55.0 28.0

$oluble

* .f

Y54.0 f

4.9 2.3

1.5 mM

407.0 356.0 cumene

of 1.O ml 10 mM

hydroperoxide

phosphate

buffer,

(I mM ANIT. a-naphthylisothiocyanate: 30 min at 37°C’. The nuclear proteins, 2, Materials and Methods. in Section * S.D. of !hrcr to four experiments.

(CHP)

2.8 15.2 5.7

l

f or

pH 7.5. Some

I mM

NADPH.

of the reactions

I mM OA. n-octylamine histones. NH2 and NH30 were Binding of [‘HIDES to various

D. Roy t-t al. /Cancer

higher covalent binding of [‘HIDES to all three types of nuclear proteins (1037 f 88, 695 f 28, 3288 f 481 pmol/mg protein/30 min for histones, NH2 and NH30, respectively) than that observed in the presence of NADPH (141 f 4, 115 f 11 and 954 f 28 pmol/mg protein/30 min for histones, NH2, and NH30, respectively) (Table I). Higher binding during cumene hydroperoxidedependent reaction was possible because only oxidation of DES to DES quinone will occur. Reduction of DES will not be possible because cumene hydroperoxide is not a cofactor of cytochrome P450 reductase 161. Whereas during the NADPHdependent nuclear activation of DES, both oxidation of DES to DES quinone and reduction of DES quinone to DES were possible, which might have produced lower binding of [3H]DES to nuclear proteins. Addition of known cytochromes P450 inhibitor ANIT, OA, or SKF-525A [6-81 either in cumene hydroperoxide- or NADPHdependent reaction inhibited the covalent binding of [3H]DES to all three types of nuclear proteins. The inhibition of binding ranged between 43-95%. We also examined the binding of [3H]DES to nuclear proteins of kidney nuclei in the presence of cumene hydroperoxide or NADPH. Like hepatic nuclei, the binding of [3H]DES in the presence of

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90 (1995)

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cumene hydroperoxide was higher in all three types of nuclear proteins of nuclei from kidneys (416 f 78, 520 f 28 and 1411 f 52 pmol/mg protein/30 min for histones, NH2 and NH30, respectively) than that observed in the presence of NADPH (158 f 21, 167 f 11 and 315 f 23 pmol/mg protein/30 min for histones, NH and NH30, respectively) (Table 2). Addition of known cytochromes P450 inhibitor OA [6-81 either in cumene hydroperoxide or NADPH-dependent reaction inhibited the covalent binding of t3H]DES to all three types of nuclear proteins. The inhibition of binding to nuclear proteins by OA ranged between 42-68% (Table 2). It is concluded that [3H]DES in the absence of co-factor cumene hydroperoxide or NADPH is not able to irreversibly or covalently bind to nuclear proteins. [ 3H]DES reactive metabolites formed in the presence of liver or kidney nuclei and cofactor bind to nuclear proteins. This reaction is presumably nuclear cytochromes P450dependent. 3.2. [“HIDES quinone-RNA polymerase or DNA polymerase I adducts

We also investigated the site of attack on pure

Table 2 In vitro binding of [3H]DES to histones, nonhistones precipitable by 2% TCA (NH2) and nonhistones soluble in 2% TCA (NH30) in the presence of hamster kidney nuclear activation system Conditions

Binding of [3H]DES to nuclear proteins (pmohmg protein/30 min) Histones

NH2

NH30

416.5 zt 7.8 132.5 * 3.9

520.0 f 9.7 240.0 b 5.8

1411.0 l 52.2 340.0 f 43.8

158.5 zt 2.2 33.2 zt 2.5

167.5 f. 10.6 105.0 f 5.6

315.0 f 23.2 180.0 zk 24.7

CHP as a cofactor

Complete system +OA NADPH

as a cofactor

Complete system +OA

The reaction mixture consisted of 10 mg purified hamster kidney nuclei, 1.5 mM cumene hydroperoxide (CHP) or I mM NADPH, 100 PM DES (4000 counts/min/pmol [)H]DES) in the final volume of 1.0 ml 10 mM phosphate buffer, pH 7.5. Some of the reactions were also carried out in the presence of cytochromes P450 inhibitor, 1 mM n-ocylamine (OA). Reaction mixtures were incubated for 30 min at 37OC in a shaking waterbath. Various nuclear proteins, histones, NH2 and NH30 proteins were isolated [8,13] and washed with organic solvent as described in Section 2, Materials and methods. Binding of [3H]DES to various nuclear proteins is expressed as pmohmg protein/30 min f S.D. of three to four experiments.

Table 3 Effect of various thiols and thiol modifiers on the binding of f3H]DES quinone to pure nonhistone protein c’onditions

pmol 13H]DES quinone boundmg RNA pal

pmol i3H]DES quinone bound/mg DNA pal -I-

Complete system

155.0

192

f

7.1

f

ed that [3H]DES quinone was irreversibly bound to RNA polymerase (155 i 7.2 pmol DES quinone bound/mg RNA polymerase protein) and DNA polymerase I(192 f 21 pmol DES quinone bound/ mg protein) (Table 3). Preincubation of RNA polymerase or DNA polymerase with thiol modifier: j3-hydroxy mercuric benzoate, n-ethylmaleimide or dithionitrobenzoic acid resulted in the inhibition of binding of 13H]DES quinone to both RNA and DNA polymerases (Table 3). The inhibition of binding ranged between 35-82%. When the binding of t3H]DES quinone to polymerases was evaluated in the presence of low molecular weight thiol, GSH, it was observed that GSH inhibited the binding to RNA polymerase and DNA polymerase by 93 and 81%, respectively (Table 3). Cysteine inhibited binding to RNA polymerase and DNA polymerase by 71 and 68%, respectively (Table 3). We have shown that the turnover of [3HJDES quinone in water soluble fraction in the presence of GSH or cysteine was higher than that in the absence of thiol [18]. Based on these data it appears that the sulfhydryl group of DNA or RNA polymerase is probably one of the sites of attack by DES metabolites. The inhibition of [3H]DES quinone binding to polymerase may be a result of the interaction of GSH, cysteine, or thiol modifiers with [3HJDES metabolite

21

Thiols

+CSH +Cysteine Thiol

11.0 f 1.8 46.0 f 2.6

37.0 f 61.0 f

4.0 1.6

mod$ers

+NEM 55.0 f 3.9 35.0 f 2.1 +PHMBA 36.0 f 2.0 58.0 f 3.4 47.0 f 2.2 68.0 f 5.0 *-DTNB __ The reaction mixture consisted of 50 pg pure RNA polymerase or DNA polymerase I, IO0 pM thiol (GSH or cysteine) or thiol modifier (I mM N-ethytmaleimide, NEM: 0.1 mM phydroxymercuricbenzoic acid, PHMBA; dithionitrobenzoic acid, DTNB), 100 gM (3H]DES q&one (8000 counts/mini pmol) in the tinal vo!ume of 1.0 ml 10 mM phosphate buffer. pH 7.5. Reaction mixtures were incubated for 60 min at 24°C. Protein was extracted, precipitated and washed as described in Section 2, Materials and methods. Binding of [‘HIDES quinone to protein is expressed as pmol/mg proteimmin *SD t,f three to four experiments.

nonhistone proteins RNA polymerase and DNA polymerase by [3H]DES quinone, a reactive metabolite of DES. Incubation of RNA polymerase or DNA polymerase I with 13HJDES quinone reveal-

!ii,yl

--, .-I, A ?,.: _

‘; !!

2

K!I

30

Ftg. 2. In vivo binding of [3H]DES to nuclear protiens of the target organ of cancer (kidney) and nontarget organs (liver and testis). Each hamster received an i.p. injection of 50 mdkg DES (containing 2S &i [3HJDES). After 4 h animals were killed, liver, kidney and testis were excised. The nuclei were isolated according to the procedure of Blobel and Potter [IO]. The histones, NH2 and NH30 were isolated from each organ as described in Section 2, Materials and methods. Free [‘HIDES and its metabohtes were removed [7,8,14]. The values shown represent the mean of five to six experiments, i SD. Cross-hatch. liver; vertical hatch, kidney; horizontal hatch. testis

D. Roy et al. /Cancer

Lerrers

90 (1995)

221

215-224 I

making it unavailable for binding to the -SH groups in polymerase is not ruled out.

+

3.3. In viva binding

The binding of [3H]DES to nuclear proteins was identified in the nuclei of target organ of cancer (kidney) and of nontarget organs (liver and testis) of hamster treated with a single i.p. injection of 50 mg/kg DES (187 rmol/kg containing 25 &i) [3H]DES) (Fig. 2). The levels of [3H]DES binding to kidney histones, NH2 and NH30 were 26, 40, 1250 pmol/mg protein, respectively. The level of binding to all three types of nuclear proteins in both non-target organs (liver and testis) was similar. When the binding was compared with target organ vs. nontarget organ, it was observed that the level of [3H]DES binding to all three nuclear proteins (histones, NH2, NH30) in the kidney (target organ) was several fold (two or more) higher than that observed in the liver or testis (Fig. 2). The pattern of in vivo binding of DES to liver nuclear proteins compared to that of kidney was opposite of what we observed for in vitro binding of DES to nuclear proteins of hepatic nuclei in the presence of cofactor compared to that of kidney nuclei (i.e. higher binding in hepatic nuclei vs renal nuclei). The exact reason for differences in the level of binding between in vivo and in vitro is not clear. However, it is known that both metabolizing and detoxifiying enzymes are higher in liver compared to that of kidney [7]. Besides. differences in repair and dilution of binding due to protein synthesis, in vivo availability of steady state concentration of DES and DES binding metabolites in both liver and kidney may be responsible for higher in vivo binding of DES to kidney nuclear proteins compared to that observed in hepatic nuclear proteins. The maximum in vivo binding occurred in NH30 proteins compared to histones and/or NH2 proteins of nuclei from all three organs (liver, kidney and testis), which is similar to that observed in vitro. The in vivo binding appears to be irreversible or covalent in nature because the radioactivity coprecipitated with nuclear proteins and could not be extracted with organic solvents. To confirm this, NH30 proteins bound to [3H]DES were subjected to SDS-PAGE and gel slices were counted

wfds

Radioac!ivity

(DPM) +

56

<21

744 -.-

37


892

33


358

28

<25

383

Fig. 3. SDS-PAGE of 2% TCA soluble nonhistone proteins (NH30) from the nuclei obtained from the kidneys of hamster treated with [3H]DES (187 pmolikg) (+) and untreated hamster kidney nuclei (-). The NH30 proteins were isolated from the nuclei of untreated and DES treated hamster kidneys [13]. Free [3H]DES and its metabolites were removed by organic solvents extraction [7,8,14]. The NH30 proteins were resolved on 15% polyacrylamide gel. The position of molecular weight marker proteins is indicated by solid line. Molecular weight of [3H]labeled nonhistone proteins is shown by arrow. The radioactivity present in the corresponding nonhistone protein bands of (-) and (+) lanes is shown in bottom panel.

to determine the distribution oi [‘HIDES bound :o NH30 proteins. It was observed that four prc! tein bands of approximate molecular weights .56 i7. 33 and 28 kDa (Fig. 3) were radioactive (744. X92. 35X. 3X3 dpm, respectively). Proteins of rni~. lecular weight 56 and 37 kDa were more radioar~, tive than the other protein species. Protein band:: of the same molecular weight from nuclei reactet! with [‘HIDES in the absence of cofactor showed radioactivity equivalent to background r90-- .Jr! dpm). incubation of [3H]DES with pure histom or HMGs 1 and 2 did not show any incorporatiori of radioactivity (data not shown). Micrococcal nuclease preferentialI> ;ittackx. chromatin regions containing trdnscrihed DNII !16.19] and the rate of attack is sensitive to gcnltranscription rate. It releases transcriptionally do. tive chromatin in macromolecular form and wr!! allow a direct measure of DES binding I(, :KX:V chromatin components. We fractionated i!l 1:;) (: bound [‘HIDES-bound chromatin b:i mlcrococcai nuclease digestion procedure [ 1hj. which IL+~~c$ :: supernatant fraction containing actively transcrihed DNA sequences and engaged RN A polymerast. 11, Covalent binding of [‘HIDES trj NHP.10 pro teins i$ supernatant fraction i*btainc(i t’ron! micrococcal nuclease trealed renal nuclc: cvilb I ICO pmolimg protc.ii.;

incubation of kidney nuciei with 3) or lO0 g.?n DES revealed an ~nhib!tor:, rftcc: on 1.n~ tc>t:ti RNA polymurase actlvit! 111the present;: oi enci(b gcnous template (Table 41. ‘The percent Inhibitlo:? ranged from 7.?~-.38’:~~~. No mhibltlon Ott ir:mscr~~. IIon activity by DES was observed in isolated TIUJei in the presence of exogenouslq added DNA template. Incubation of nuclei with DES in the presence OF NADPH (shown 10 convert DES I(; nuclear protein binding metaholites) [Sj
TJhk 4 LnhihirLrn

0i transcrlprional

activity

by DES

metabolism pmol

[ ‘?P]GTP

incorporated: mg DNA ___..- .._

-__-._-._--._-_--_-_~-~.~~----Nucicl or Nuclei t NADPH Nuch .( DES (50 +M) Nuclet Yuck!

+ DES t- DES

(100 !I00

I I mM)

/LM,I pM1 +, Y\DPH

._..... ..__....__._ -.-_-_-... ----.-_The reaction mixture consisted (IO&-125 gg DNA), 5U mM Iris-HCI.

il of

mM) hamster pH 7.9,

41.0

f

2 8

31.0 25.0

f *

1.6 2.1

12.0

f

0 8

.._.-.---. kidney nuclei 2 mM MnCl,.

300 mM (NH.,@04, 200 &I .i’ NTPs and I pc(‘i [u-i’P]GTP I‘he reactton mixture was Incubated with DES, NADPH or DES plus NADPH for 10 or 30 min prior of starting reaction 0~ adding ia-“P]CiTP u-Amanitin was added at a final concentratIon

of 20 &ml or 200 rrspctiveiy.

RNA pkii 111. awa\ured h! ‘he

method

p&ml to inhibit 1 ranscriptional

RNA

pol activll!,

II

and wa$

of’ Yu et al. [IT].

4. IGassion We have demonstrated for the first time that DES was converted in an in vivo system to nuclear the level ot protein binding metabolite(s); :‘H]DES binding to all three types of nuclear proteins (histones. NH?. NH30) in the kidney (target ;>rgan) was se\:eral fold (two or more) higher than that observed in the liver or testis (nontarget organs); four nuclear NH30 proteins (mol. wts. 56. ;\-I. 33 and 28 kDa) were irreversibly bound to I-‘HIDES in Gvo: activation of DES to nuclear protein binding metabolites by nuclei appears to hc cytochromes P450 dependent: sulfhydryl group ot‘ nonhistone proteins may be one of the sites of attack by [‘HIDES metabolites; and [‘HIDES hinds to transcriptionally active chromatin proteins and inhibits transcriptional activity. In vivo modification in the transcription regulating proterns by DES metabolires may influence gene functior!. Recently WC have shown direct evidence of rat hepatic nuclear cytochromes P450-catalyzed redox cycling of DES [6]. During the redox cycling, DES is oxidized to DES quinone, presumably via DES semiquinone. by nuclear cytochromes P450 (i.e. in the presence of nuclei and cumene hydroperoxide):

D. Roy et al. /Cancer

DES quinone is reduced back to hydroquinone by nuclear cytochrome P450 reductase, presumably via semiquinone. Direct evidence of intermediacy of DES semiquinone is yet to be shown. Binding of DES to hepatic or renal nuclear proteins observed in this study is dependent on the avaibility of cofactor, cumene hydroperoxide or NADPH. This binding was inhibited by cytochromes P450 inhibitors. These findings suggest that hamster hepatic or kidney nuclei also are capable of metabolizing DES to nuclear protein binding reactive metabolites, and this reaction appears to be nuclear cytochromes P450 dependent. Chromosomal proteins, histones and nonhistones, provide structural support to the chromatin, as well as play an important role in several aspects of gene function [20,21]. Chemical modifications in nonhistone proteins have been shown to influence accurate reading of DNA template during transcription or replication [20]. Therefore, it is logical to suggest that modification of nuclear proteins involved in the control of gene function by DES reactive metabolites may influence gene activity. As observed in this study that transcriptionally active chromatin proteins are susceptible to the in vivo attack by DES reactive metabolites, and DES metabolites inhibit transcriptional activity, provide support to this concept. The binding of DES metabolites to histones or nonhistones could stimulate or block an essential site for biochemical modification of nucleosomes during changes in gene function. The influence of modification to nuclear proteins by DES metabolites could be long lasting, since the turnover of some of the nuclear proteins is low and there is a lack of repair of damage to polypeptides [20,21]. Moditication of histone and nonhistone proteins by electrophilic metabolites of carcinogens has been implicated to participate in chemical carcinogenesis [9,20]. DES causes renal cancer in hamsters [l]. We have recently observed that treatment of hamsters with a carcinogenic dose of DES produced an inhibition of transcriptional activity in vivo [22]. It is possible that in vivo modification chromatin proteins by DES reactive metabolites may play a role in DES-induced renal carcinogenesis, however, if remains to be elucidated.

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Acknowledgements

This research was supported by the NIH grant (CA52584). References

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