[CANCER RESEARCH 48, 2462-2468, May 1, 1988]

Quantitative Structure-Activity Studies on Effects of Sixteen Different Steroids on

Growth and Monooxygenases of Rat LiverR. Schuhe-Hermann,1 H. Ochs,2 W. Bursch, and W. Parzefall

Institut fürToxikologie und Pharmakologie der Philipps-Universität, Pilgrimstein 2, D-3550 Marburg Federal Republic of Germanyand Institut fur Tumorbiologie-Krebsforschung der UniversitätWien, Borschkegasse 8a, A-1090 Wien, Austria

ABSTRACT

Sixteen steroids with different endocrine activities were administeredto female rats for 6 or 7 days, in a broad range of doses. Liver growthwas recorded by measuring weight and DNA contents and monooxygenaseactivity by assaying the turnover of five different substrates. Accordingto their effects on these parameters steroids were assigned into one ofthe following three groups: (a) Estrogens estradiol and ethinylestradiol,as well as the progestins norethynodrel and norethisterone (norethin-

drone) which have estrogenic activity in rats. These agents inducedpronounced liver growth and excessive DNA increase which was notassociated with major monooxygenase induction, (b) A different type ofresponse consisted of liver growth and DNA increase associated with apronounced induction of monooxygenase(s) in a characteristic pattern.This response was elicited by pregnenolone-16 «-carbonitril, by proges

tins progesterone, cyproterone acetate, and medroxyprogesterone (butnot gestoden and levo-norgestrel), by the antimineralocorticoid spirono-

lactone and by the glucocorticoids cortisol and dexamethasone. Apparently, this response pattern was not related to any specific endocrineaction but to certain structural features, in particular to the presence ofa saturated, at least two-membered alkyl substituent at C17 of the steroidring system, (c) No or small effects were observed after gestoden, levo-

norgestrel and the androgens testosterone and methyltestosterone.Dose-response studies revealed that estrogens estradiol and EE2 in

duced hepatic effects more potently by four orders of magnitude thanprogestins. The response patterns observed may be relevant to the tumor-

promoting activity of some of the steroids tested.

INTRODUCTION

Long-term use of contraceptive steroids in rare cases mayresult in tumor appearance in human liver. Such tumors usuallyseem to be benign and in some instances regressed after withdrawal of steroids (1-3). No evidence of genotoxic or tumor-initiating activity of the steroids has been detected (3-7). It hastherefore been assumed that tumor appearance in human liverunder contraceptive medication is due to a promoting effect onthe growth of accidental preneoplastic lesions.

Experiments on rodent animals have clearly supported thepossibility of a tumor-promoting action of certain contraceptivesteroids. This was found with both estrogens (8-11) and progestins (12, 13). So far it is not known whether the promotingactivity is due to specific endocrine or structural properties ofthe steroid molecules. However, in recent years evidence hasaccumulated suggesting that many liver tumor promoters sharethe ability to induce growth in this organ. This has beenobserved with progestins (14, 15) and estrogens (16, 17) andalso with nonsteroid tumor promoters such as phénobarbital,hexachlorocyclohexane, dichlorodiphenyltrichloroethane, po-lychloriuated biphenyls carbazine, tetrachlorodibenzo-p-dioxin,etc. (18, 19). Liver growth observed after application of these

Received 7/20/87; revised 12/23/87; accepted 1/28/88.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 inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1To whom requests for reprints should be addressed, at Institut fürTumorbil-ogie-Krebsforschung der Universität Wien, Borschkegasse 8a, A-1090 Wien,Austria

2Present address: Universitäts-Frauenklinik, Albert-Schweitzer Strame 33, D-

44 Münster,Federal Republic of Germany.

agents does not appear to occur as a response to tissue damageas such damage is usually absent. On the other hand, livergrowth frequently is associated with functional increases suchas of drug-metabolizing monooxygenases and is therefore considered an adaptive response.

The apparent association of liver tumor promotion withgrowth and monooxygenase induction in this organ made itworthwhile to investigate whether any relation may exist between these hepatic actions and structural or endocrine properties of the steroids. Therefore we have studied the effects of16 different steroids with diverse chemical structures includingestrogens, gestagens, antiandrogens, glucocorticoids, and anantimineralocorticoid (Fig. 1). Where feasible several doses ofthe steroids were tested for quantitative comparisons of theirefficacy and for estimation of NOEL.3 Some results obtained

with single steroids have already been published (14-16, 20,21). In this paper we present a compilation of the observationsmade with all of the 16 agents. The results suggest that according to their hepatic effects the steroids can be grouped intothree different classes which may be distinguished by endocrineand structural properties.

MATERIALS AND METHODS

Animals and Treatment. Female Wistar rats (SPF) were obtainedfrom Zentralinstitut fürVersuchstierzucht, Hannover, Germany. Theywere 7-9 weeks old and weighed 140-180 g at the start of treatment.Animals were housed five per cage in a climatized room under controlled light-dark rhythm (lights on from 0900-2100, off from 2100-0900). In a few experiments these lighting conditions were somewhatmodified as follows: continuous lighting, Figs. 3 (F:.) and 5 (progesterone); inversed light-dark rhythm, Fig. 2 (EE2, CPA) and Figs. 3 and 5(PCN). Various control experiments have shown that these modifications have no significant effects on the results obtained. A powdered orpelleted standard rat chow (AItromin 1321; Altrogge, Lage, Germany)was provided ad libitum. Tap water was always available.

All steroids investigated were obtained from Schering AG (Berlin,Germany). The identity and purity of the agents had been analyticallyconfirmed; purity was greater than 98% in all cases. At least five ratswere used for each control or treatment group. For s.c. treatment agentswere dissolved in castor oil:benzylbenzoate (3:2). Concentrations wereadjusted to inject maximally 2 ml vehicle/kg. CPA was dissolved incorn oil (0.4%) to administer 10 ml/kg, PCN was suspended in water;both agents were administered orally by gavage. Control animals received pure vehicle or remained untreated, no significant effect of thevehicle on the parameters studied was found (22). Other steroids to begiven orally were premi ved with lactose and then admixed to powdereddiet. Food consumption was measured daily, and the steroid doseapplied was calculated. Animals were treated for 7 days at the onset ofthe light phase, killing by decapitation was at the same time on Day 8.The liver was quickly excised, blotted, and weighed. Microsomes wereprepared from fresh liver; specimens for other biochemical assays werestored at —15°C.Liver samples for histológica! investigations were

fixed in formalin (4%).

3The abbreviations used are: NOEL, no observed effect level; AP, aminopyrine,AN, aniline; BPA, benzphetamine; CPA, cyproterone acetate; E2, estradiol EE2,ethinylestradiol; EM, ethylmorphine; LI, labeling index; PNA, p-nitroanisole;PCN, pregnenolone-16a-carbonitril.

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STRUCTURE-ACTIVITY STUDIES ON HEPATIC EFFECTS OF STEROIDS

Estradici Ethinylestradlol

OH

C=CH

Norethynodrel (Morethisterone—3061316

QCOCH3CECH

Progesterone Cyproterone -

acetate

OH - Cyproterone -

acetate

Spironolactone

Medroxyprogesterone -

acetate

Pregnenolone -

16*-carbonitrlleCortisol Dexamethasone

Testosterone Methyltestosterone Levonorgestrel Gestoden

Fig. I. Structures of compounds investigated.

Biochemical Assays of Liver Composition. Measurements for DNA,RNA, and protein are as follows. Liver specimens were homogenizedin 10 vol cold 2% perchloric acid with 0. l MEDTA. After centrifugaronthe sediment was washed twice in cold 2% perchloric acid. DNA andRNA were extracted into 5% perchloric acid at 80°Cfor 15 min. DNA

was then determined according to Burton (23), and RNA according toFleck and Munro (24). Where required the 3H content of the extracts

was assayed in a liquid scintillation counter, and calculated as dpm/MgDNA. Protein was measured as described by Lowry (25).

Mk rosoinal Enzymes. Liver specimens from each treatment groupwere pooled and homogenized in 5 vol saccharose (0.25 M), buffered topH 7.4 with tris. The microsomal fraction was obtained by di^rentialcentrifugal ion at 10,000 and 100,000 x g, and was stored prior to useat —15°C.This does not affect enzyme activities as shown in separate

experiments. Incubation mixtures contained in a final volume of 0.5ml: NADP (5 x 10~4M), isocitrate (5 x 10~3M), isocitrate dehydrogen-ase (20 mU), MgCl2 (3 x 10~3M), and one of the following substrates:AP (10~2 M), EM (5 x 10~3M), BPA (10~3 M), AN (2 x 10~3M), pNA(0.5 x 10~3). All substances were dissolved in phosphate buffer (pH

7.4, M/15). The samples were incubated for 20 min in a shaking waterbath at 37°C.Enzyme reaction was stopped by transfer into an ice bath

and addition of trichloroacetic acid (1.8 M, 25 (/I). Formaldehydeformation from AP, EM, and BPA was measured according to Nash(26), pNA 0-demethylation was determined by measuring p-nitro-

phenol formation (27) and AN metabolism by measuring p-amino-phenol formation (28). All assays were conducted in triplicate.

Histológica!Procedures. Liver specimens were embedded in paraffin.Sections, 5-prn thick were cut, stained with hematoxylin-eosin, andautoradiography was performed with Kodak foto emulsion NTB3. Thepercentage of labeled cells (LI) was determined for hepatocytes (LIhepatocytes) and for sinus wall cells (LI sinus wall cells); the percentageof hepatocytes in mitosis was also determined. At least 2000 hepatocytes were counted in each liver.

Statistics. Means obtained from five rats and standard deviations aregiven. The significance of differences was checked by Student's / test.

Some of the results are expressed in percentage of controls from therespective experiments for the purpose of clarity. Statistical significanceof steroid-induced changes was always checked against the controlsfrom the respective experiments using the absolute data. A correctionfor multiple application of the test method to the same data (e.g.,Bonferoni correction) was not performed.

RESULTS

Liver Growth. Of the series of steroids tested the estrogenEE2 and the progestin CPA were found to be the most potentinducers of liver growth. Effects of these two agents were

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STRUCTURE-ACTIVITY STUDIES ON HEPATIC EFFECTS OF STEROIDS

therefore investigated in greater detail and are displayed forcomparison in Fig. 2. 0.5 mg/kg EE2 and 40 mg/kg CPA wereadministered s.c. and orally, respectively. As shown EE2 increased liver size by 21% and liver RNA and DNA by 33%;CPA enhanced these three parameters by approximately 40%(Fig. 2,). Both agents stimulated hepatic DNA synthesis asshown by biochemical and autoradiographic determination of[3H]thymidine uptake into DNA and nuclei, respectively (Fig.

2). The pronounced increases of hepatocyte LI and mitoticactivity (Fig. 2) indicate that parenchymal hyperplasia is animportant factor in the overall liver enlargement. In additionsinus wall cells showed an enhanced LI and therefore seemedto participate in the growth process (4, 6). It should also benoted that both agents increased DNA synthesis only transiently; apparently, at the end of the treatment period of 7 and6 days, respectively, liver size and DNA approached a newsteady state at an enhanced level. In summary, we concludefrom these findings that both EE2 and CPA induce orderedgrowth in female rat liver.

In further studies we determined liver mass and DNA asparameters representative of liver growth and hyperplasia. The

Liver weight4g/100g

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STRUCTURE-ACTIVITY STUDIES ON HEPATIC EFFECTS OF STEROIDS

Fig. 3. Relative liver weight and DNA content of the liver after treatment with varioussteroids. •.liver weight; I"J,liver DNA. Col

umns show the increases observed in steroidtreated rats in percentage of control animals.Control values (= 100%) were: relative liverweight: 4.09 ±0.28g, DNA 12.8 ±1.8 mg/OOg,OOg.Significant differences from controlanimals: *, p < 0.05; »*,p < 0.01; ***, p <

0.001.

Fig. 4. The influence of hydrocortisone anddexamethasone treatment on body and livergrowth. Dexamethasone (/') 130 mg/kg oncedaily, hydrocortisone (//«) SO mg/kg oncedaily, hydrocortisone (H.,,,,) 200 mg/kg oncedaily. U. body weights before treatment; 0,after treatment; columns; horizontal lines andshaded areas, controls (means ±range of SD).

140

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STRUCTURE-ACTIVITY STUDIES ON HEPATIC EFFECTS OF STEROIDS

Ethinylestradiol

0,5 mg/kg.s.c.

Norettlynodrel

lOOmg/kg.s.c.

Norethisterone -aœlale50mc/kg,s.c.

nnÃ-lnnAP EM BFUAH

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-500

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480mg/kg,orally 50mg/kg,s.c. SOmgAg.s.c.200mc/kg,s.c.•-ni

inninn1IIII1 IInnium11PiinnMedroxyprogesterone - Pregnenolone - Cortisol Dexamethasoneacetate i6«-carbonitrile200 mg/kg.s.c. tOOmg/kg.orally 200 mg/kg.s.c. 130 mg/kg.s.c.

300 - n

Methyttestosterone Levonorgestrel200 mg/kg,s.c. 5mg/kg,s.c.

Gestodene mg/kg.orally

3-Melhylcholantrene Phénobarbital200 mg/kg.orally lOOmg/kg.orally

Fig. 5. Patterns of monooxygenase activities. Doses indicated were given oncedaily for 6 or 7 days. Columns indicate the turnover of test substrates by pooledmicrosomes in ^g product/mg microsomal protein x 20 min expressed as apercentage of controls. Control values were: AP, 1.57 ±0.45; EM, 0.76 ±0.28;BPA, 1.19 ±0.30; AH, 1.10 ±0.27; pNA, 1.75 ±0.53; Results with PCN, 3-MC, and PB are taken from Ref. 20.

dose-dependent enhancement of EM turnover. The NOEL asestimated by extrapolation was approximately 0.002 mg/kg.Enzyme induction after progesterone and CPA treatment wasmuch more pronounced, and clearly dose-dependent. NOELswere approximately 30 mg progesterone/kg and 4 mg CPA/kg,respectively. The different doses of levonorgestrel did not exertany effect on EM demethylation. Gestoden showed a tendencyto increase the activity of EM-demethylating enzymes.

DISCUSSION

We conclude from this study that the steroids investigatedcan induce at least two different types or patterns of responsein the liver as summarized in Table 1: (a) Liver growth char-

200

100

300

200

100

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7o200 •Levonorgestrel-3

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n-TT""nun-3-2 -1 0nJl-Õ1II12 3

log dose (mg/kg)

Fig. 6. Ethylmorphin demethylation: Dose response relations with differentsteroids. Columns, turnover of EM by pooled microsomes from steroid treatedanimals in percentage of controls. Control values were 1.01 ±0.20 /ig HjCO/mgmicrosomal protein/20 min.

Table 1 Summary of hepatic responses to the steroids testedExtents of increases over controls as obtained with the maximally effective

doses are indicated in an approximate way by 0-+++.

Liver growth Enzyme pattern like

Response typeorgan enlargement DNA increase PCN EE2

IEstradiolEEjNorethynodrelNorethisterone

IIPCNProgesteroneMedroxyprogesteroneCPAOH-CPASpironolactoneCortisolDexamethasone

IIIGestodenLevonorgestrelTestosteroneMethyltestosterone

acterized by an excess of relative DNA increase over weightincrease, associated with no or small monooxygenase increasesof the "EE2 pattern." (¿>)Liver growth with relatively smaller

or no increases of DNA, associated with pronounced monooxygenase increases of the "PCN pattern"; dose-response kinetics

increased more steeply than in group 1. We have assigned thesteroids to one of these groups. Steroids with small or no effectson liver growth and monooxygenases that could not unequivocally be assigned to groups 1 or 2 were assigned into a thirdgroup (type III) (Table 1).

Can these response types be correlated to any known endocrine or structural properties of the steroids? Group 1 comprisesthose four steroids of the present study that in rats act predominantly and strongly estrogenic at the effective doses (29).Consequently, we hypothesize that the responses of group 1may be due to estrogenic activity of the agents. This conclusionis supported by inhibitory effects of clomiphene, an antiestro-gen, on estrogen-induced liver growth and DNA synthesis (17,

30).In contrast, group 2 responses do not appear to be correlated

with any particular endocrine effect: PCN has virtually no2466

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STRUCTURE-ACTIVITY STUDIES ON HEPATIC EFFECTS OF STEROIDS

endocrine effects, progesterone, medroxyprogesterone, CPAand hydroxy-CPA are progestins, spironolactone is an amimineralocorticoid, and cortisol and dexamethasone are glucocor-ticoids. On the other hand, progestins levonorgestrel and ges-toden cannot be assigned to group 2. It is, however, remarkablethat the steroids of group 2 share structural properties whichare not found in groups 1 and 3, i.e., a saturated alkyl substituent with at least two carbon atoms at position 17 (Fig. 1).Apparently, this side chain may be hydroxylated at C2i or not,may be substituted at C!7 in a (spironolactone) or ß(othersteroids) configuration, and can be part of a lactone. However,unsaturation of the side chain as in ( ', -cihinyl derivatives

(groups 1 and 3) seems to cause inactivity.All three types of response of the steroids investigated clearly

differ from those of phénobarbitalor methylcholanthrene. Thisobservation is consistent with the results of previous studies(20, 31, 32).

In most earlier investigations hepatic effects of single or onlya few steroids were reported. The results generally agree withthose of the present work, e.g., induction of liver growth byestrogens in the absence of pronounced stimulation of monoox-ygenase activities (3, 33, 34). Testosterone and methyltestoster-one did not induce distinct liver enlargement (35) but, unlikeour observations, methyltestosterone had a distinct inducingeffect on EM demethylation (36, 37); the reason of this discrepancy is not known. Induction of liver growth and/or of mon-ooxygenases by PCN, CPA, spironolactone, medroxyprogesterone, and other members of our group 2 was also noted before(20, 32, 38-40).

An extensive structure-activity study with respect to monoox-ygenase induction was reported by Heuman et al. (31). Theseauthors used an immunological assay to show that hepaticconcentrations of the PCN-inducible cytochrome P45oincreasedfollowing treatment of rats with spironolactone, cortisol, anddexamethasone, but not after E2, mestranol, testosterone, andmethyltestosterone. In vitro studies using isolated hepatocytessuggested that induction of P450-PCN is mediated by a receptormechanism unrelated to the "classical" glucocorticoid receptor

(41). While these results strongly support our conclusion as toexistence of group 2 of steroid effects, Heumann et al. (31)found no increase of cytochrome P4so-PCN in rat liver in vivoafter progesterone. This apparent difference from our study isprobably due to route of application and dosage of progesteroneused by those authors (50 mg/kg i.p. once daily) (see 31). Sinceprogesterone has a short biological half-life this regimen maynot provide liver levels sufficiently high for a sufficient periodof time to induce monooxygenase(s).

With respect to liver tumor promotion it is of interest to notethat most promoting steroids recognized by now would beclassified in group 1 or 2 of the present investigation, i.e.,estradiol and estradici esters (8), EE2 (9-11, 42), CPA, progesterone (12,13), cortisol, and dexamethasone (42). Testosteroneslightly enhanced the number (but not the size) of 7-GT positivefoci in rat liver (42); a combination of testosterone esters hadno significant effect on liver tumor development in intact ratsbut was promoting in gonadectomized rats (43). Thus testosterone may have a weak tumor-promoting effect in rat liver,and this would be paralleled with the present observations wherethe compound although assigned to group 3 did produce someliver growth. In summary these observations seem to supportour hypothesis that the ability to induce liver growth is animportant property of hepatic tumor promoters, although astrict quantitative correlation between stimulation of livergrowth and tumor promotion does not appear to exist (44, 45).

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