[CANCER RESEARCH 56. 4391-4397. October 1. 1996]

Steroidal Drug Cyproterone Acetate Is Activated to DNA-binding Metabolites

by SulfonationSilke Werner, Susanne Kunz, Thomas Wolff, and Leslie R. Schwarz1

GSF-Foi-sctnínxs-entritnt fürUmwelt itnil Gesundheit. Institut fürTo.\ikoi(it>ie, D-85764 Netiiierberfi/Mnnich. GYrmi/m

ABSTRACT

The antiandrogenic and gestagenic steroid cyproterone acetate (CPA)has been widely used in human therapy. There is currently a debate aboutthe safety of CPA, since it proved to he genotoxic in rat liver and humanhepatocytes |I. Neumann et al., Carcinogenesis (Lond.l, 13: 373-378, 1992;J. Topinka et al., Carcinogenesis 11.onil.i. 14: 423-427, 1993; L. R.

Schwarz et al.. Biological Reactive Intermediates: V. Basic MechanisticResearch in Toxicology and Human Risk Assessment, pp. 243-251, 1996;A. Martelli et al, Carcinogenesis (Lond.), 16: 1265-1269, 1995].

Little is known about the metabolic pathways of activation of CPA togenotoxic metabolites. Using rat hepatocytes and subcellular fractions offemale rat liver, we have examined whether sulfoconjugation plays anessential role in the activation of CPA to DNA-binding metabolites whichare detectable with '"P-postlabeling. Incubation of hepatocyte cultures

with 30 /UMCPA for 6 h caused the formation of several DNA adducts; thetotal adduci level amounted to about 12,400 adducts/109 nucleotides.

When the cells were incubated in sulfate-free medium to prevent thesynthesis of the cosubstrate of sulfonation, 3'-phosphoadenosine-5'-phos-

phosulfate (PAPS), formation of all CPA-DNA adducts was greatly re

duced, amounting to only 5% ofthat determined in the presence of sulfate(810 /nM). Activation of CPA is likely to be catalyzed by hydroxysteroidsulfotransferase(s), because the specific substrate dehydroepiandros-terone almost completely inhibited DNA-binding of CPA. Our assumption

that sulfonation plays a decisive role in the bioactivation of CPA is furthersupported by the results obtained with an in vitro system consisting of calfthymus DNA, various subcellular liver fractions, and the cofactor PAPS,NADPH, or NADH. Significant DNA binding only occurred when cytosoland both PAPS and the reduced pyridine nucleotides were present. TheDNA adduct spot obtained was chromatographically identical to theadduct spot A detected in isolated liver cells, suggesting that the CPA-

DNA adduct formed in vivo and in vitro is identical. Cytosol is known tocontain not only sulfotransferases but also reductases. Thus, the requirement for NADPH or NADH suggests that in addition to sulfotrans-

ferase(s), reductases are involved in the activation of CPA.We propose that bioactivation of CPA involves reduction of the keto

group at C-3 followed by sulfonation of the hydroxysteroid. The resulting

sulfoconjugate is most likely unstable and supposed to generate a reactivecarbonium ¡on.

INTRODUCTION

The antiandrogen and gestagen CPA2 is used in the treatment of

acne, hirsutism, prostate carcinoma, and to inhibit sexual drive insexual deviants. In particular, Diane and Diane-35. which contain

CPA in combination with ethinyl estradiol. have obtained widespreaduse as antiacnegenic drugs with contraceptive activity. Similar toother synthetic sex steroids, the progesterone derivative has beenshown to induce liver tumors in rats (5). Tumorigenicity of the steroidin rats has been attributed to tumor promotion. This notion is supported by the finding that the steroid shares several biological activ-

Receivcd 1/3/96: accepted 8/1/96.The costs of publication of this article were defrayed in part by the payment of page

charges. This article must therefore be hereby marked advertisement in accordance with18 U.S.C. Section 17.14 solely (o indicale this fact.

1To whom requests for reprints should be addressed."The abbreviations used are: CPA. cyproterone acetate (6-chloro-17ur-acetoxy-l,2a-

methvlenepregna-4.6-diene-3,2()-dione): DHEA. dehydroepiandrosterone; PAPS. 3'-phosphoadenosinc-S '-phosphosulfate; TES. ¿V-tris(h)droxymelhyl )melhyl-2-aminoeth-

anesulfonic acid.

¡tieswith other known liver tumor promoters in the rat, such as theinduction of growth of the liver and preneoplastic hepatocytes (1,6-9), a decrease in apoptosis (10), and induction of cytochrome P450

(9), and by the lack of genotoxicity of CPA in standard in vitromutagenicity tests (11, 12). However, our laboratory has recentlypresented evidence that CPA has not only tumor-promoting but alsogenotoxic and tumor-initiating activity. This evidence is based on the

following findings: (a) CPA induces DNA repair and the formation ofDNA adducts in liver cells of rat and man (1-3. 13, 14); (è)persistentCPA-DNA adducts are formed in the rat (2, 15); and (c) the synthetic

steroid initiates the formation of preneoplastic hepatocytes in femalerats when tested in the rat liver foci bioassay (16).

The genotoxicity of CPA is characterized by two special features inthe rat: it is largely restricted to the liver (2, 3) and it exhibits a markedsex difference, female rats being much more sensitive to the genotoxicaction than male rats (2). In view of the fact that CPA did not bind tocalf thymus DNA in the absence of subcellular fractions in vitro (2),these findings collectively suggest that CPA is activated to DNA-reactive intermediates by drug-metabolizing enzymes of the liver

which are sex specific. However, the pathway of the activation ofCPA is unknown. Preliminary experiments indicated that cytochromeP450 does not play a major role in the generation of DNA-binding

metabolites from the steroid (3). Another metabolic route which couldlead to activation of CPA may involve reduction of the steroid at C-3(17) and subsequent sulfonation of the 3-hydroxy derivative. The

sulfoconjugate will most likely be unstable, giving rise to a reactivecarbonium ion. In the present investigation, we therefore studied thepossible involvement of sulfoconjugation in the activation of CPAusing isolated hepatocytes and subcellular fractions.

MATERIALS AND METHODS

Biochemicals. Spleen phosphodiesterase and proteinase K were purchasedfrom Boehringer Mannheim (Mannheim. Germany): CPA. DHEA. PAPS,micrococcal nuclease. potato apyrase (grade VI). RNase T, (R 1003) fromSigma (Deisenhof'en. Germany); calf thymus DNA and RNase A from Serva

(Heidelberg. Germany); T4 polynucleotide kinase from Amersham (Frankfurt.Germany); poiyethyleneimine-cellulose TLC sheets, 0.1 mm. from Macherey-Nagel (Duren. Germany); and [•y-<2P]ATP.tetratriethylammonium salt (spe

cific activity. 3(KX)Ci/mmol) from NEN-DuPont (Dreieich. Germany).Animals. Female Wistar rats (8-10 weeks old. inbred strain; Neuherberg)

were fed a standard diet (Altromin; Lage) and had free access to tap water.Isolation and Culturing of Hepatocytes. Rat hepatocytes were isolated by

collagenase perfusion of the liver (24). except that the perfusion media did notcontain sulfate. Viability of the cells was routinely determined by staining withtrypan blue. More than SOVrof the hepatocytes excluded the dye.

Hepatocytes (7 x 10'' viable cells in 7 ml) were seeded into collagen-coated

100-mm dishes. A modified DMEM was used which excluded L-cysteine,L-methionine, and sulfate (MgSO4 X 7H,O was replaced by MgCU X 6H,O)and was supplemented with 10 ITIMHEPES, 10 mM TES, 0.1 juM dexametha-

sone. 5 milliunits insulin, and, during the short attachment period, with 10ng/ml epidermal growth factor. Cells were allowed to attach for l h at 37°Cin

a humidified atmosphere of 59c CO2 in air. After changing the medium,incubation was continued in the presence of 30 ¡JLMCPA and various concentrations of MgSO4 X 7H2O or DHEA. CPA was dissolved in DMSO. The finalconcentration of DMSO in the medium was 0.25% (v/v). After incubation for3 h or 6 h. the cells were washed twice with 10 ml ice-cold PBS. scraped from

the plates, and transferred into tubes used for DNA isolation.

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ACTIVATION OF CYPROTERONE ACETATE

Bioactivation of CPA in Vitro. CPA (50 /¿M)was incubated in the theassay buffer with calf thymus DNA (0.2 mg/ml) and facultatively with livercytosol (4 mg protein/ml). S9-mix (liver homogenate obtained after centrit'u-

gation at 9000 X g; 4 mg protein/ml), liver microsomes (4 mg protein/ml).PAPS (0.2 min). NADH (0.25 mM), and NADPH (0.1 mM) at 37°Cfor 1 h. The

assay buffer consisted of KH,POj (1.5 min). Tris (10 mM), KC1 (150 mM),MgCl: x 6H,O (2 mM), EDTA (0.2 mM), and 5'-AMP-Na2 (0.5 mM) and wasadjusted tu pH 7.0 at 37°C.Glucose-6-phosphate (5 mM) and glucose-6-

phosphate dehydrogenase (2 units/ml) were added as components of aNADPH-regenerating system. To test whether both NADPH and NADH can

function as reduction equivalents in the metabolism of CPA. the regeneratingsystem was omitted and replaced by 2 mM NADPH or 2 mM NADH. Cytosol.59-mix. and microsomes were prepared according to Remmer et al. (19) with

the following modifications. The homogenization solution (250 mM sucrose)was supplemented with DTT (0.1 mM) and KH2PO4 (10 mM; pH 7.2 at 4°C).

Microsomes were washed with KC1 (150 mM). DTT (0.1 mM). K2HPO4 (10mM), and EDTA (10 mM). Cytosol was dialyzed three times against 2 liters ofhomogenization solution for about 6 h at 4°C.The repetitions of the experi

ments were performed with independent preparations of the subcellular fractions.

Isolation of DNA from Hepatocytes and in Vitro Incubations. Hepato-

cytes were scraped into I ml of extraction buffer consisting of Tris (IO mM).EDTA (0.1 M), and SDS (0.5%; pH 8.0). After the addition of 10 /tl RNase A( 10 mg/ml) and 10 /¿IRNase T, (4000 units/ml), the suspension was incubatedfor 2 h at 37°C.Then the proteins were digested by proteinase K (0.4 mg/ml)at 37°Cfor 2 h. DNA was extracted with phenol and subsequently with

chlorotbrm/isoamylalcohol (24:1, v/v). DNA was precipitated by the additionof 100 /xi NaCl (5 mM) and 1 ml of absolute ethanol. The DNA pellet waswashed twice with 707c ethanol. Finally. DNA was dissolved in a SSC solutionconsisting of NaCl (10 mM) and sodium citrate (10 mM; pH 8.0).

In vitro incubations were stopped by the addition of phenol. Extraction ofDNA was performed as mentioned above. The precipitates were kept at -20°C

until resuspension in 500 /¿ITESSC buffer (20 HIMTris. 1 mM EDTA, 10 mMsodium citrate, and 10 mM NaCl. pH 8.0). After incubation with 15 /il RNaseA (10 mg/ml) and 15 /xl RNase T, (4000 units/ml) for 30 min, the sampleswere treated with 0.25 tng/ml proteinase K at 37°Cfor another hour. Extrac

tion, precipitation, and washing of DNA were performed as described above.Measurement of DNA Adducts. DNA adducts were determined according

to the procedure of Gupta (20). as described recently (15). To compare theadduci spots detected on the autoradiographs performed with DNA of in vitroincubations and cultured hepatocytes, both samples were chromatographedwith a modified solvent system as well. In the modified system. 0.6 Mammoniawas used in direction D2 instead of 3.6 M lithium formiate and 8.5 M urea (pH3.5) to achieve a higher resolution in the cochromatography experiments.

All values were corrected for the background level determined on a controlchromatogram at the corresponding position. The DNA digest used for thesecontrol chromatograms was derived from hepatocytes incubated only with thesolvent DMSO. For the determination of cpm in total nucleotides. aliquots ofthe DNA hydrolysate were highly diluted, labeled in the presence of an 11-foldexcess of [32P]ATP. and chromatographed on polyethyleneimine-cellulose

sheets with 80 mM ammonium sulfate. The spots at the start region containingthe normal and modified nucleotides were excised, and the radioactivity wasdetermined by Cerenkov counting. Water blanks were run for correction.Relative adduci labeling was calculated as described by Gupla and Dighe (21):(cpm in adduci nucleotides/cpm in total nucleotides) x (I/dilution faclor).

RESULTS

Studies with Hepatocyte Cultures. Incubation of the hepatocytesof female rats with 30 juM CPA caused the formation of several DNAadducts. The radiochromatograms of the 32P-postlabeled nucleotides

(DNA fingerprints) showed eight adduci spots not detectable in chromatograms from control incubations (Fig. 1, a and c). The DNAadducts were tentatively designated as A-H. Adducts A-D were chro-

matographically identical to the adducts A and D, which we havedescribed recently (2, 15). Because of improvements in the 32P-

postlabeling method, higher DNA adduci levels were detected in thepresent study than previously (2, 15) and additional minor DNA

H

Fig. 1. Autoradiograms of ^P-postlabeled DNA adducls from CPA-lrealed hepalocyte

cultures of female rats. Liver cells were incubated in sulfate-containing medium (SII) ¡UÀ)for 6 h with 0.25% (v/v) DMSO (a), with 30 /IM CPA in sulfate-free medium (b). or with30 /AMCPA in sulfate-containing medium (r). Adducts were purified, posilaheled with"P. and separated as described in "Materials and Methods." Exposure was 2 h. A-H,

adducts.

adducts, i.e., adducts E-H, became visible. Incubating the cells for 6 h

with the synthetic steroid attained total DNA adduci levels of about12,400/109 nucleotides. Similar to previous studies, DNA adduci A

was by far predominant and adduci D was the second most frequent;the levels of these two adducts amounted to about 11.000 and 400/1 Oy

nucleotides, respectively (Table 1).To study the possible involvement of sulfoconjugation in the acti

vation of CPA to DNA-binding metabolites, hepatocytes were incubated in sulfate-free medium. Sulfate is required for the biosynthesis

of the cofactor of sulfonation, PAPS. The medium used in theseexperiments was free of inorganic sulfate and the amino acids methi-

onine and cysteine, which may undergo oxidative metabolism toinorganic sulfate (22-24). As shown in Figs, le and 2, formation ofCPA-DNA adducts in hepatocytes was strongly dependent on the

presence of sulfate in the medium. In the absence of sulfate, totalDNA binding of CPA amounted to only 5% of the binding determined

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ACTIVATION OF CYPROTERONE ACETATE

at the normal sulfate concentration of 810 JU.M.At a concentration of25 /U.Msulfate in the medium, almost maximum CPA-DNA adduci

formation was attained (Fig. 2, note that the X axis is interrupted). Amore detailed analysis of the effect of sulfate on the levels of theindividual CPA-DNA adducts shows that formation of all DNA

adducts detected in the hepatocyte cultures strongly depends on thepresence of sulfate in the medium (Table 1).

Of the various sulfotransferase isoenzymes catalyzing sulfonationof endogenous and foreign compounds, hydroxysteroid sulfotrans-

ferase(s) may possibly catalyze sulfation of CPA. We therefore performed cell incubations in the presence of DHEA. DHEA and DHEA-

sulfate. which is most likely formed in hepatocytes, are known toselectively inhibit hydroxysteroid sulfotransferase(s) (25). As shownin Fig. 3. DHEA strongly inhibited the formation of CPA-DNAadducts in hepatocytes. Half-maximal inhibition is already attained at20 /J.M,and at a concentration of 100 JU.MDHEA. almost no CPA-

DNA adducts were formed (Fig. 3).In Vitro Studies. To further substantiate the role of sulfoconjuga-

tion in the activation of CPA and to study the involvement of reduction in the formation of reactive metabolites in addition to sulfonation,activation of CPA has been studied in vitro using subcellular fractionsof the livers of female rats and cofactors. The in vitro systemscontained calf thymus DNA and the following components in variouscombinations: cytosol or S9-mix as a source of sulfotransferases and

Table I Effect of sulftitf (in the formation of DNA atldui-ls by CPA in l

cultures (tf female rats

DNAadduci"''1AHCI)EFGHRelativeadduci1Sulfate-free574

*99n.d'n.d.''20

±9n.d.''n.d.''2±

1n.d.''abeling'

(X109)Sulfate

(810(¿M)10.809•¿�*-1.26215*423

±5388±9921*723

±5118±386

±2" Letters indicate the individual DNA adducts formed by CPA.' Hepalocytes were incubated with 30 ^xMCPA in sulfatc-free and sulfate-containing

medium (810 (¿M).1 Results represent the mean ±SD of three independent experiments.'' n.d.. not detectable.

O

X

14000 -

12000 -

10000 -

8000 -

6000 -

4000 -

2000 -

0 -

5 10 15 20 25 800 1000

MgSO4 •¿�7 H20

Fig. 2. Dependence of the formation of CPA-DNA adducts in hepatocytes on inorganicsulfate. Hepatocyte cultures of female rats were incubated with 30 [JLMCPA in mediacontaining various sulfate concentrations for 6 h. Adducts were purified, postlabeled with'2P. and separated as described in "Materials and Methods." (Note that the X axis ¡s

interrupted.) Results represent the means of three independent experiments. Bars, SD.

Coo

100 -

80

60

40

20

O -—¿�i—

20

—¿�i—

40

—¿�i—

60

—¿�i—

80 100

Dehydroepiandrosterone

Fig. 3. Effect of DHEA on the formation of DNA adducts by CPA in hepaiocytecultures of female rats. Hepatocytes were incubated with 30 JIM CPA and with variousconcentrations of DHEA in sulfate-containing medium (SIO /IM) for 3 h. Adducts werepurified, postlabeled with 1:P, and separated as described in "Materials and Methods."

Results represent the means of three independent experiments. 1009Õ- 5454 ±465

adducts/1 (f nucleotides. Burs. SD.

reductases, and the cofactors PAPS. NADH, and NADPH( + NADPH-regenerating system). Under the in vitro conditions, onlyone DNA adduci was detectable (Fig. 4). This adduci was chromato-graphically identical to adduci A, the predominant DNA adduci de-

lermined in hepatocytes (Fig. 5). Similarly, the two adducts showedidentical retention times using high-performance liquid chromatogra-phy.3 As shown in Fig. 6, significant DNA binding only occurred

when the incubations contained both PAPS and reduced pyridinenucleotides in addition to cytosol or S9-mix. Additional experiments

showed that NADPH can be replaced by NADH. In the presence of 2IBMNADPH or 2 m.MNADH (and absence of the NADPH-regener-

ating system), DNA binding amounted to 568 ±20 and 688 ±63(n = 2), respectively. Bioactivation of CPA was prevented when thecytosol was denatured by heating to 95°Cfor 15 min (data not shown).

Similarly, formation of CPA-DNA adducts was insignificant when thecytosol or S-9-mix was replaced by microsomes. In the presence ofmicrosomes, reduced pyridine nucleotides, the NADPH-regenerating

system, and PAPS. DNA binding of CPA amounted to only about0.239?- ±0.07% (n = 3) of that determined ¡nthe presence of liver

cytosol and the cofactors. The requirement for reduced pyridinenucleotides in the generation of DNA-binding metabolites suggests

the involvement of reductase(s). which is present in the cytosol of theliver cell.

DISCUSSION

Sulfonation plays a key role in a number of essential biologicalpathways, including biotranslbrmation of steroidal hormones, xeno-

biotic detoxification, and carcinogenic activation (compare Ref. 22).Transfer of SO, from the sulfonate donor PAPS to endogenous andforeign compounds is catalyzed by various sulfotransferases. Duringthe past four years, several c-DNAs of sulfotransferase have been

cloned, such as phenol sulfotransferases. hydroxysteroid sulfotransferases, and estrogen sulfotransferase (26-29). Several sulfotrans

ferases have overlapping substrate specificities. For a long time,sulfonation has been known to play an important role in the activationof several xenobiotics. The first electrophilic metabolite of a carcin-

' B. Binkova. personal communication.

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ACTIVATION OF CYPROTERONE ACETATE

Fig. 4. Autoradiograms oí'~P-posllabcled DNA ad-

ducts from in ri/m incubations. Cylosol or S9-mix (4 mgprotein/mil. NADH (0.25 niMI. NADPH (0.1 mM), theNADPH-regencraling system, and PAPS (0.2 mw) wereincubated in the presence of 0.25% (v/v) DMSO or 30 JIMCPA lor 1 h. Adducts were purified, postlabeled with l~P.and separated as described in "Materials and Methods." u.

cytosol/DMSO; b. cytosol/CPA; c. Sv>-mix/DMSO: and </.SM-mix/CPA. I-xposure was 6.5 h.

ogen to be discovered was the sulfoconjugate 2-acetylaminofluorene-¿V-sulfate(30. 31 ). Since then several carcinogens have been shown tobe metabolically activated by sulfonation, e.g.. safrol. 4-aminoazo-benzene, and numerous benxylic alcohols of polycyclic aromatic-

hydrocarbons (22, 32-34). The sulfonates of these carcinogens are

unstable, and elimination of the sulfate results in an electrophilicnitrenium ion or carbonium ion which can bind to DNA.

In the present investigation, we show that sulfonation is involved in

Fig. 5. Autorudiograms of chromatography and cochro-matography of ''P-postlabeled DNA adduci A troni hepa-

tocyles and ihc DNA adduci formed in vitro. In a, liver cellsfrom temale rais were incubated with 30 p.M CPA lor 6 h;2 jug DNA were used for the autoradiography. In h. the invitro incubation was performed with cylosol. NADH/NADPH, and PAPS as described in the legend of Fig. 4; 10/u.g DNA were used, c, mix of samples used in a ( l /¿gDNA) and b (5 /¿gDNA). The solvent system for all threechrornatograrns differed from that used in Fig. 1 and 4 toobtain u higher resolution (see "Materials and Methods").

Exposure was 2 h.

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ACTIVATION OF CYPROTERONE ACETATE

the bioactivation of the synthetic steroid CPA. This is evident from themarked dependence of CPA-DNA adduci formation in hepatocytes on

inorganic sulfate and the requirement for cytosol (sulfotransferases)and PAPS in vitro. Half-maximal formation of CPA-DNA adducts is

observed even at the low sulfate concentrations of 15 /UM.Sulfonationof CPA is most likely mediated by hydroxysteroid sultbtransferase(s)(29, 35). as indicated by the inhibition of CPA-DNA adduci formation

by DHEA, which is a substrate of hydroxysteroid sulfotransferases(36).

Involvement of hydroxysteroid sulfotransferases in the activation ofCPA would be in line with our previous findings, which showed thatthe genotoxicity of CPA was largely restricted to the liver and wasmuch higher in female rats than in male rats (2, 3). Similarly, certainhydroxysteroid sulfotransferases have been shown to be preferentiallyexpressed in the liver and to exhibit much higher activity in the liversof adult female rats as compared to male rats (36, 37). In contrast tohydroxysteroid sulfotransferases, expression of aryl sulfotransferaseIV, which bioactivates iV-hydroxy-2-acetylaminofluorene, and of es

trogen sulfotransferase is much higher in male rat liver (38, 39). Theactivity of rat hepatic hydroxysteroid sulfotransferase toward andros-

terone has been shown to exhibit characteristic alterations duringpostnatal development (37). It increased after birth in both sexes untilthe weanling stage, thereafter decreasing in males. Compatible withthis developmental pattern, we found high CPA-DNA adduci levels inthe liver of male rats when 21-day-old animals were treated with CPA

(40).In contrast to hepatocytes of adult female and male rals, similar

DNA adduct levels have been determined when human hepatocytes ofboth sexes were incubated with CPA (14). This finding would also becompatible with the involvement of hydroxysteroid sulfotransferase inthe activation of the synthetic steroid, since expression of hepaticDHEA-sultbtransferase has been reported to be similar in women and

men (41).Finally, involvement of liver-specific hydroxysteroid sulfotrans

ferase would also explain the lack of genotoxicity in standard in vitromutagenicity tests in the presence of S9 and NADPH (12). External

r TOO-Om

E3

E'xO)

E

<tr

80 -

60 -

40 -

20 -

NADIPIH + - +

PAPS - - + +

Fig. 6. Formation of DNA adducts by CPA ¡nvitro, dependence on the presence ofcytosol or S9-mix and cofactors tor reduction and sulibnation. CPA (30 JIMwas incubatedwith cytosol or S9-mix (4 mg protein/ml) in the presence or absence of NADH (0.25 mw).NADPH (O.I mm), the NADPH-regenerating system, and PAPS (0.2 niM). Adducts werepurified, postlaheled with KP. and separated as described in "Materials and Methods."

Results represent the means of three independent experiments. Bars, SD. Cytosol.100% = 2308 ±429 adducts/10g nucleolides: S9-mix. 100% = 864 ±206 adducts/10g

nucleotides.

reduction

CPA

elimination

Fig. 7. Proposed metabolic pathway of the activation of CPA by sequential reductionand sulfonation of the steroid at C-3. It is suggested that a reactive carbonium ion is

formed by elimination of the sulfate group.

metabolic activation in these test systems did not include sulfonationdue to the lack of the cofactor PAPS. Even if reactive sultoconjugateswere to be formed externally, mutations may not necessarily beinduced in the indicator cells since sulfoconjugates may be short-lived

or not be able to cross the membrane of the indicator cells (42).The present in vitro studies suggest that, in addition to sulfonation.

reduction of CPA represents an essential step in the bioactivation ofthe steroid, since binding to calf thymus DNA only occurred in thepresence of cytosol/S9-mix, PAPS, and NADPH/NADH. Thus, wepropose that the keto group of CPA at C-3 is first reduced and

subsequently sulfuted. giving rise to a reactive sullbconjugute (Fig. 7).This assumption is supported by preliminary data from our laboratoryshowing that CPA is efficiently metabolized to the 3-hydroxy steroidin the in vitro system used.4 Moreover, Kerdar er cd. (17) recently

showed that 3-a-OH-CPA is a major metabolite of CPA in rat bile and

suggested that this metabolite is involved in the bioactivation of thesteroid. Up to now several keto reductases/3-hydroxysteroid dehydro-

genases have been identified in rat and human liver cytosol andendoplasmic reticulum (43). The present results suggest the involvement of cytosolic reductase(s); it is not yet clear to what extent

4 Unpublished results.

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ACTIVATION OF CYPROTERONE ACETATE

reductases of the endoplasmic reticulum may also take part in thebioactivation of CPA.

In the in vitro experiments, only one DNA adduci was detectable,which is likely to represent adduci A, the most predominant adduci inhepatocytes. The reason for the lack of the other adducts determinedin the intact cell system is not clear. One may speculate that thecombined action of reducíase, sulfotransferase, and additional drug-metabolizing enzymes such as cytochrome P450-dependent mo-nooxygenases may give rise to ine adducts B-H which are deleclablein hepatocytes. In the presence of PAPS, NADPH, and S9-mix (cy-tosol + endoplasmic reticulum), however, these adducts were notdetectable.

It is inleresling lo note thai ine proposed palhway of aclivation ofCPA has clear analogies lo one of ihe pathways leading to theactivation of the antiestrogen tamoxifen and the synthetic stilbeneestrogen diethylstilbestrol. Recently, it has been reported that hy-droxylalion al ihe allylic a-carbon of ine ethyl side chain of thesemolecules is most likely followed by sulfonation to ultimate DNA-

damaging metabolites (44, 45).The present study indicates that sulfonalion by hydroxysleroid

sulfotransferase(s) plays a major role in the bioactivation of CPA. Weput forward the hypothesis lhat CPA is reduced to the 3-hydroxy

derivative prior to the formation of a reaclive sulfoconjugate (Fig. 7).Following elimination of Ihe sulfate anión,an electrophilic carboniumion will be formed. This reaction will be facilitated, since the conjugated double bonds delocalize the positive charge, thereby slabilizingihe carbonium ion. At present it is not clear whether sulfate elimination proceeds via a SN1 or a SN2 mechanism.

ACKNOWLEDGMENTS

We are very grateful to Dr. Jan Topinka for performing the cochromatog-raphy experiment and to Dr. Blanka Binkova for the high-performance liquid

chromatography analysis.

REFERENCES

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2. Topinka. J.. Andrae. U. Schwarz. L. R., and Wolff, T. Cyproterone acetate generatesDNA adducts in rat liver and in primary rat hepatocyte cultures. Carcinogenesis(Lond.), 14: 423-427. 1993.

3. Schwarz. L. R.. Werner, S., Topinka. J.. Andrae. U.. Neumann. !.. and Wolff, T. Theliver as origin and target of reactive intermediates exemplified by the progesteronederivative. Cyproterone acetate. In: R. Snyder J. J. Kocsis. I. G. Sipes, G. F. Kalt". D.

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4. Martelli. A.. Mattioli. F.. Fazio, S., Andrae. U.. and Brambilla. G. DNA repairsynthesis and DNA fragmentation in primary cultures of human and rat hepatocytes exposed to Cyproterone acetate. Carcinogenesis (Lond.), 16: 1265-1269.

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1996;56:4391-4397. Cancer Res   Silke Werner, Susanne Kunz, Thomas Wolff, et al.   Metabolites by SulfonationSteroidal Drug Cyproterone Acetate Is Activated to DNA-binding

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