Effect of pretreatment of male syrian golden hamsters with 7,8-benzoflavone and with diethylstilbestrol on P-450 isoenzyme activities and on microsomal diethylstilbestrol metabolism

0022-473 1,‘SS$3.00+ 0.00 Pergamon Press plc

.I. steroid &o&em. Vol. 31, No. 6, pp. 971-978, 1988 Printed in Great Britain

EFFECTOF PRETREATMENT OF MALE SYRIAN GOLDEN HAMSTERS WITH 7,8-BENZOFLAVONE AND WITH DIETHYLSTILBESTROL ON P-450 ISOENZYME ACTIVITIES AND ON MICROSOMAL DIETHYLSTILBESTROL METABOLISM G~NTER BLAICHand MANFRED METZLER Institute of Pharmacology and Toxicology, University of Wilrzburg, Versbacher Strasse 9, D-8700 Wibzburg, F.R.G.

(&ceiued 3 March 1988)

Summary-Combinecl treatment of male Syrian golden hamsters with the synthetic estrogen diethylstilbestrol (DES) and 7,8-benzoflavone (7,8-BF) gives rise to a high incidence of hepatocellular carcinomas, whereas no such tumors are formed with DES alone nor with 7,8-BF alone. To determine whether alterations in DES metabolism may account for the observed hepatocarcinogenicity, we have studied the effect of pretreatment with 7,8-BF alone, DES aione and 7,8-BF plus DES on the levels of hepatic P-450 and cytochrome bs, on the activities of various P-450 isoenzymes and on microsomal DES metaboiism. Hepatic P-450 content was significantly increased after pretreatment with 7,8-BF and decreased after DES, while combined pretreatment led to levels similar to those in untreated control animals. Hepatic cytochrome b, was also elevated in 7,bBF-treated hamsters; DES pretreatment had no effect, and combined pret~atment ied to a slight increase. Four different substrates were used to probe P-450 isoenzyme activity. Aryl hydrocarbon hydroxylase (AHH), 7-ethoxycoumarin-0-deethylase (ECOD), 7-ethoxyresorufin-0-deethylase (EROD) and 7-pentoxyresorufin-0-dealkyla~ (PROD) were all elevated after 7,8-BF-pretreatment, while DES led to a decrease in these activities with the exception of AHH, where a transient increase which was observed after 8 and 20 weeks of pretreatment was back to control levels after 32 weeks. Combined pretreatment with 7,8-BF and DES led to an intermediate response (slight increase) with AHH, EROD and PROD, but not with ECOD, where a full induction comparable with that observed after 7,8-BF alone was elicited. In spite of the modulation of enzyme levels and activities observed after the various pretreatments, the metabolism of DES in microsomes from pretreated animals was virtually identical with that from controls. Therefore it is concluded that modulation of hepatic DES metabolism is not the reason for the observed hepatotumorigenicity; instead, it is speculated that 7,8-BF is the carcinogenic agent in this tumor model, and DES may act as a promotor.

INTRODUCTION

The synthetic estrogen diethylstilbestrol (DES) is carcinogenic to animals and humans [ 11.Male Syrian golden hamsters chronically exposed to DES develop renal tumors with a 100% incidence [2]. There are several lines of evidence suggesting that oxidative metabolism of DES generates reactive inte~~iates similar to those formed in the metabolism of classical chemical carcinogens 131. These electrophilic intermediates may play a crucial role in the development of renal tumors in the hamster [4-61. Various oxidative pathways have been demonstrated, but at present it is not clear which one is important for the tumorigenicity of DES; it is quite conceivable that different pathways are critical for the carcinogenic effect in different tissues. Formation of the clear cell carcinoma in the hamster kidney was inhibited by treatment with vitamin

C or other antioxidants [7,8]. Combined treatment with DES and 7,&benzoflavone (7,8-BF), an inhibitor of NADPH-dependent monooxygenases [9]* also reduced renal tumors in male hamsters significantly but resulted in the induction of multinodular hepatic tumors [lo]. This pronounced shift in the organ-directed tumo~genesis of DES coutd be explained if 7,8-BF alters the metabolism of the estrogen in such a way as to affect the carcinogenic activity of the hormone. The consequence of the altered metabolism of DES by 7,8-BF might be a decrease in the concentration of reactive intermediates in the kidney and an increase in the liver. Only few studies are as yet available on the modulation of DES metabolism by 7,8-BF pretreatment [3, 111. Analysis of the pattern of urinary metabolites showed that untreated animals excreted mostly hydroxylated metabolites and little Z,Z-DIES, whereas 7,8-BF pretreated hamsters pro971

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BLAICH and MANFRED METZLER

duced smaller amounts of hydroxylated compounds and increased amounts of Z,Z-DIES [3]. In vitro studies with liver microsomes from hamsters pretreated with 7,8-BF or DES for 3 days, on the other hand, exhibited no marked changes in the pattern of DES metabolites compared with untreated controls [12]. In the present study we have investigated whether the metabolism of DES in male hamster liver microsomes is affected by pretreatment with 7,8-BF alone, with DES alone, or with a combination of 7,8-BF and DES. The study should help to clarify the role of metabolic activation of DES in its mechanism of carcinogenicity. EXPERIMENTAL

Materials

[2-14C]DES (sp. rad. 53 mCi/mmol, The Radiochemical Centre, Amersham, U.K.) was shown by HPLC to be 95% radiochemically pure, and to consist of 81% Eand Z-isomer. 19% Z,Z-dienestrol (Z,Z-DIES, Z,Z-3,4-his-(p-hydroxyphenyl)-hexa-2,4-diene) and I-hydroxy-Z,Z-DIES were prepared in our laboratory as previously described [13]. Unlabelled E-DES and 4’-hydroxypropiophenone were purchased from Merck (Darmstadt, F.R.G.). I-hydroxy-E-DES and 3’-hydroxyE-DES were kindly provided by Dr John A. McLachlan (National Institute of Environmental Health Sciences, Research Triangle Park, U.S.A.) and Z-DES by Dr P. Murphy (Lilly Research Laboratories, Indianapolis, U.S.A.). NADP+, NADH, NADPH, sodium dithionite, sodium isocitrate and benzo(a)pyrene were from Serva GmbH (Heidelberg, F.R.G.). Isocitrate dehydrogenase and 7-pentoxyresorufin were from Boehringer (Mannheim, F.R.G.). 7,8-BF (97% purity), 7-ethoxycoumarin and 7-hydroxycoumarin were purchased from Aldrich Chemie (Steinheim, F.R.G.). Resorufin and 7-ethoxyresorufin were from Molecular Probes Inc. (Eugene, U.S.A.). 3’-hydroxybenzo(a)pyrene was kindly provided by Dr W. Wiebel (Gesellschaft fiir Strahlenforschung, Neuherberg, F.R.G.). All other chemicals, reagents and solvents were of analytical grade. Animals

Male Syrian golden hamsters (S&l00 g b. wt) were obtained from the Zentralinstitut fur Versuchstierzucht (Hannover, F.R.G.). The animals had access to pellets of laboratory chow (Altromin 1324, Altrogge, Lage/Lippe, F.R.G.) and tap water ad libitum. They were kept in groups of 5 under controlled conditions of temperature and humidity on a 12-h light, 12-h dark cycle and acclimated at least 4 weeks prior to use. *According clature [29].

to

the

newly-recommended

nomen-

Pretreatment of animals

For pretreatment with 7,8-BF, the hamsters received a pelleted laboratory chow of Altromin 1324 containing 0.4% 7,8-BF ad libitum. This diet was prepared by Altrogge (Lage/Lippe, F.R.G.). Daily food consumption was 5-7 g, resulting in a daily dose of about 200 mg/kg b. wt. For pretreatment with DES a 20 mg pellet was implanted S.C.in the shoulder region. An additional pellet was implanted every 3 months in order to maintain tissue levels. Preparation of microsomes

The hamsters were killed by cervical dislocation. The liver was perfused with 0.9% saline containing 1 mM EDTA through the portal vein, removed, rinsed and placed on ice after removal of the gall bladder. Homogenization was carried out at 4°C in a Potter-Elvehjem vial for 2 x 15 s at 400 rpm in a buffer of pH 7.6 containing 25 mM HEPES, 1.5 mM EDTA, 1 mM DTT, 100 mM NaCl and 10% glycerol. The homogenate was centrifuged at 4°C and 10,000 g for 20 min and the postmitochondrial supernatant centrifuged at 4°C and 105,OOOgfor 60 min. The microsomal pellet was resuspended by gentle homogenization in the same buffer and centrifuged again under the same conditions. Finally, the pellet was suspended in the buffer to yield a concentration of 20-25 mg microsomal protein/ml. Protein was estimated according to Lowry et a1.[14]. Enzyme activity assays

Total P-450* was determined from the dithionitereduced carbon monoxide difference spectra. Cytochrome b5 was measured from the NADH-reduced difference spectra as described by Omura and Sato[lS]. All spectral measurements were made with a dual beam spectrophotometer (Model Uvikon 860, Kontron, Zurich, Switzerland). Aryl hydrocarbon hydroxylase (AHH) was assayed at 37°C by the formation of 3-hydroxy-b-enzo(a)pyrene according to Nebert and Gelboin[l6]. The dealkylation of 7-ethoxyresorufin (EROD) and 7-pentoxyresorufin (PROD) was measured at 25°C by the method of of Mayer[ 171. 0-deethylation Burke and 7-ethoxycoumarin (ECOD)was assayed according to Ullrich and Weber[l8]. A model SFM-23 spectrofluorimeter (Kontron, Zurich, Switzerland) was used for the fluorimetric assays. Microsomal metabolism studies

Incubations of microsomes (1 mg microsomal protein/ml) were carried out in triplicate in a total volume of 2 ml 100 mM potassium phosphate buffer pH 7.4 containing 80 PM DES (0.3 PCi [14C]DES). After a preincubation period of 1Omin at 37°C the metabolism was started by adding a NADPHregenerating system containing 1 mM NADPH, 3.3 mM MgCl,, 7 mM isocitrate and 0.5 U/ml isocitrate dehydrogenase. In control experiments the

P-450 activities and DES metabolism

microsomes were boiled at 96°C for 10 min before adding NADPH. After 30min at 37°C with gentle shaking, the incubation was stopped by adding 100 ~1 of an aqueous 1 M citrate-l M ascorbate solution and the aqueous phase was extracted 4 times with a 2-fold volume of diethyl ether-ethyl acetate (1: 1; v/v). The combined organic extracts were concentrated to dryness under reduced pressure and the residue dissolved in 200 ~1 methanol for HPLC analysis. The microsomal protein in the extracted aqueous phase (2.1 ml) was precipitated by adding 1 ml of a 20% aqueous solution of trichloroacetic acid (TCA). After centrifugation and washing with 2 ml 5% TCA solution the precipitate was dissolved in 1 M NaOH (2 ml, 6 h at 37°C) and an aliquot was analysed for radioactivity. HPLC analysis was carried out using a Waters instrument. A 25 cm x 4 mm id. column packed with RP-18 (Zorbax ODS 5 pm, Bischoff, Leonberg, F.R.G.) was operated at 20°C with a flow rate of 1 ml/min and a linear solvent gradient (solvent A: water-methanol 8 : 2 v/v; solvent B: methanol) changing from 45 to 100% B in 30 min. The eluate was monitored continuously with an U.V. detector at 254 nm and collected in 0.3 ml fractions. Radioactivity was measured in a model 3390 liquid scintillation counter with automatic external standardization (Packard Instruments, Frankfurt, F.R.G.). For analysis by GLC-mass spectrometry the HPLC fractions were evaporated to dryness under reduced pressure and derivatized with O,N-bis(trimethylsilyl)acetamide. GLC-mass spectrometry was performed on a Finnigan 4510 model as previously described [ 121. Statistical evaluation

Results are expressed as mean values f standard deviation (SD). Data were analyzed by Student’s t-test. P < 0.01 was considered to be significant. RESULTS

Eflect of pretreatment on P-450 and cytochrome b, levels in liver microsomes

P-450 and cytochrome b, levels were measured in microsomes from male Syrian golden hamsters pretreated with 7,8-BF, DES or DES plus 7,8-BF for 8, 20 and 32 weeks (Fig. 1). In order to account for a conceivable age-dependent alteration in enzyme activities, animals of various ages up to 45 weeks were used as controls, but were all found to have very similar enzyme levels and also activities. In the treated animals, 7,8-BF and DES were not withdrawn prior to microsome preparation in order to mimic the in vivo situation of tumor formation. In one experiment, 7,8-BF was withdrawn 48 h before sacrificing the animals: enzyme levels, enzyme activities and DES metabolism were the same as in animals without withdrawal (data not shown). Treatment with 7,8-BF alone increased both P-450 and cytochrome b, levels, both of which remained

913

Cylochrcmrs

0

0

(nmd / mg protein)

8

20 Perwad of prcircatmmt

32 (weeks)

Fig. I. Time-course of P-450 and cytochrome b, levels in hepatic microsomes from male hamsters pretreated with 7,8-BF (a), DES (A) and 7,8-BF plus DES (m). Data represent the mean k standard deviation. Number of animals: untreated 12, pretreated 4 or 5. *, P Q 0.01; *, P Q 0.001. SD for cytochrome b, levels were invariably smaller than the symbols.

elevated over the whole period of pretreatment up to 32 weeks. The relative increase in cytochrome b, content (from 0.45 to 0.8 nmol/mg protein) was greater than that of P-450 (from 1.3 to 1.8 nmol/mg protein); accordingly, the ratio of P-450 to cytochrome b, was decreased from 2.8 to 2.2. Pretreatment with DES, on the other hand, did not affect cytochrome b, content whereas the P-450 level decreased continuously with increasing time of pretreatment (Fig. 1). Pretreatment with both DES and 7,8-BF led to an intermediate effect: cytochrome b, content was only marginally increased whereas P-450 levels remained unchanged (Fig. 1). Eflect of pretreatment on various P-40-associated enzyme activities

In order to clarify whether the changes in P-450 levels are due to an increase or decrease of specific isoenzymes, the activities of ethoxycoumarin-Odeethylase (ECOD), aromatic hydrocarbon hydroxylase (AHH), 7-ethoxyresorufin-0-deethylase (EROD) and 7-pentoxyresorufin-0-dealkylase (PROD) were measured in hepatic microsomes from male hamster after the various pretreatment regimens. The results are summarized in Fig. 2. Pretreatment with 7,8-BF alone led to a significant induction of all four enzyme activities, whereas DES alone caused a moderate decrease of ECOD, EROD and PROD. Only AHH activity was increased after pretreatment with DES for 8 and 20 weeks, but returned to control values after 32 weeks. The combined pretreatment with 7,8-BF and DES had different effects on the four enzyme activities. For ECOD and AHH, there was a clear induction similar to that observed with 7,8-BF alone. For EROD and

974

G~.INTER BLAICH EC00 hol

and MANFRED MET~LER

Img x mm)

AHH hot

0

El?00hnol/mg

L, 0

f mq x min)

8

20

32

PROD @mot / mg x min 1

xmm)

I a3

a2

Period of pretreatment

(weeks)

Fig. 2. Time course of monooxygenase-associated isoenzyme activities in hepatic microsomes from male hamsters pretreated with 7,%BF (O), DES (A) and 7,8-BF plus DES (II). ECOD 7-ethoxycouma~n-O-d~thyla~; AHH, aryl hydrocarbon hydroxylase; EROD, 7-ethoxyreso~fin-Odeethylase; PROD, ?-~ntoxy~sorufin-O-deaikylase. Data represent the mean f standard deviation. Number of animals: untreated 12, pretreated 4 or 5. fi, P < 0.01; jr, P < 0.001.

PROD activities, on the other hand, the increase was much less pronounced after combined pretreatment as compared to that in 7,8-BF treated hamsters.

Microsomal metabolites of DES were separated by reverse-phase HPLC and then identified by GLC-mass spectrometry as described previously [12]. Figure 3 represents a typical HPLC chromatogram showing the U.V. absorption and the radioactivity profiles. Small amounts of radioactivity eluted very early in the chromatogram and were therefore called “polar metabolites”. The next five radioactive peaks identified as l-hydroxywere unambiguously Z,Z-DIES, 3’-hydroxy-E-DES, E-DES, 3’-hydroxyZ-DES, Z,Z-DIES and Z-DES through their mass spectra and cochromatography with reference compounds in HPLC and GLC. Radioactive material eluting as a rather broad peak at the end of the gradient was named “nonpolar metabolites” and was not further characterized. A very similar peak has been described previously in incubations of DES in

the presence of peroxidases/hydrogen peroxide and has been proposed to contain polymerization products of metabolic intermediates [191. Figure 4 summarizes the amounts of microsomal DES metabolites from untreated control animals and from male hamsters pretreated for 8, 20 and 32 weeks with DES, 7,8-BF and DES plus 7,8-BF. In order to account for a conceivable age-dependent alteration in DES metabolism, animals of various ages up to 45 weeks were used as controls, but were all found to have very similar metabolism, yielding predominantly Z-DES and the two isomers of the catechol 3’-hydroxy-DES. Minor metabolites were Z,Z-DIES and I-hydroxy-Z,Z-DIES. Pretreatment with 7,8-BF for 8 weeks marginally decreased DES metabolism whereas longer periods of pretreatment did not si~ifi~antly alter metabolism of DES as compared with controls (Fig. 4, upper panel). Pretreatment with DES for 8 and 20 weeks also did not change microsomal DES metabolism (Fig. 4, centre panel). However, treatment with DES for 32 weeks dramatically reduced DES metabolism, yielding nearly twice the amount of the parent compound E-DES and smaller

P-450 activities and DES metabolism

3

9

15

975

Zl

TimeImin)

Fig. 3. Separation of DES metabolites by HPLC. Upper chart: synthetic reference compounds; lower chart: radioactivity profile of a 30 min incubation of [‘4C]DES with hamster hepatic microsomes~

amounts of all metabolites except Z-DES. Combined pretreatment with DES and 7,8-BF for 8, 20 and 32 weeks decreased DES metabolism in the same manner: the amount of the parent compound E-DES increased about 50%, Z-DES was unaffected and all other metabolites were formed in smaller quantities (Fig, 4, lower panel).

Eflec? of pretreatment on the irreversible binding to ~~cro~~a~ prutein

The non-extractable binding of radioactivity to hepatic microsomal protein ranged from 4 to 5 nmol/mg protein x 30 min in control microsomes. After pretreatment with DES, 7,8-BF or the combination of the two, binding was decreased in all cases (Fig. 5). Whereas binding decreased continuously with microsomes from DES-treated hamsters, microsomes both from 7,8-BF and from combined treatment animals gave a decrease in binding after 8 weeks of pretreatment and no further decrease at later time points. With the exception of the &weekpretreatment, no difference could be observed between the three groups. DISCUSSION

The experimental

induction

of liver tumors

by

certain estrogens in the male Syrian golden hamster concomitantly treated with 7,8-BF has been proposed as an animal tumor model for the human liver adenomas associated with oral contraceptive use [IO]. Moreover, this animal model appears to be uniquely suited to the study of the role of estrogen metabolism in the mechanisms of estrogen carcinogenicity [lo]. If metabolic activation of an estrogen is necessary for its carcinogenic effect, the metabolic pattern should be different under conditions of tumor induction as compared with conditions which do not lead to tumor formation, We have therefore studied the metabolism of DES in liver microsomes from male hamsters pretreated for different time periods with DES alone, 7,8-BF alone, and with 7,8-BF and DES in combination. As 7,8-BF is known as inhibitor of drug metabolism, we have also looked at various P-450-associated enzyme activities in the liver. Previous studies in our laboratory have shown that DES is extensively metabolized by liver microsomes to various oxidative metabolites [ 12,201. The results of the present study are in accordance with these findings: I-hydroxy-Z,Z-DIES, Z,Z-DIES, Z-DES and the E- and Z-isomer of the catechol 3’-hydroxy-DES were unambiguously identified. An interesting feature of the in oitro metabolism not found in previous in t&o studies [11,2 I] nor in ham-

GUNTERBLAICHand MANFRED METZLER

976

Fig. 4. Pattern of oxidative DES metabolites in hamster liver microsomes after various pretreatments. Data represent percent of radioactivity applied to the HPLC column and are the mean values + standard deviation. Number of animals: untreated 10, pretreated 4 or 5. h, P < 0.01; *, P ,<0.001.

ster hepatocytes [22] is the formation of large amounts of the Z-isomer. It is known that isomerization of E-DES to Z-DES takes place under the influence of light, but this mechanism cannot account for the large quantities of Z-isomer. There is

Binding lo mcrosomal protein (nmol/rrq x30mln)

6-

0

0 Period

8

of

20

pretreatment

32 (weeks)

Fig. 5. Time-course of nonextractable binding of radioactivity to microsomal protein after a 30 min incubation of [“C]DES with hepatic microsomes from male hamsters pretreated with 7,8-BF (a), DES (A) and 7,8-BF plus DES (m). Data represent the mean f standard deviation. Number of animals: untreated 10, pretreated 4 or 5. *, P d 0.01; *, P
substantial evidence that the isomerization process is enzymatic since boiled microsomes or microsomes without NADPH are unable to form any Z-isomer (results not shown). Recent studies have demonstrated that rat liver microsomes and in particular P-450 reductase together with NADPH form Z-DES in large amounts [23]. The mechanism of the process and the enzymes involved are still a matter for debate. One possibility would be the generation of a phenoxy radical intermediate by one-electron oxidation with subsequent reduction thereby creating a redox cycle[23]. Recently it has been reported that prostaglandin synthase is able to catalyze isomerization of E- to Z-DES [24]. The effects of the different pretreatments on the levels of P-450-associated enzyme activities and of the pattern of oxidative DES metabolism were studied. 7,8-BF alone acted as a classical enzyme inducer leading to an increase in the P-450 level and in various enzyme activities. The amount of induction was comparable with that caused by known inducers such as phenobarbital (PB) or 3-methylcholanthrene (MC) and revealed that 7,8-BF induction was MClike [25]. Although 7,8-BF acted as an inducer, the metabolism of DES was either decreased or unaffected. This suggests that P-450 isoenzymes inducible by 7,8-BF are not primarily involved in DES

977

P-450 activities and DES metabolism metabolism. Pretreatment with DES alone, on the other hand, decreased P-450 content with increasing time of pretreatment. EROD activity, which represents MC-inducible &enzymes in the hamster [26], appears to be decreased to a larger extent than the other enzyme activities measured. Whether or not DES treatment specifically suppresses P-4501associated isoenzymes in the hamster liver could only be clarified by probing the isoenzymes with antibodies. Despite the reduced enzyme activities, the metabolism of DES in DES-pretreated microsomes remained virtually unaffected except after 32 weeks of pretrea~ent, at which time point all animals had large renal tumors. Under conditions of liver tumor formation, i.e. combined treatment with DES and 7,8-BF, enzyme activities showed levels resulting as a superimposition of the opposite effects of DES and 7,8-BF. Although all animals had liver tumors after 32 weeks of pretreatment, the metabolism of DES was not altered by this treatment as compared with pretreatment for a shorter time. In conclusion, the in t&o metabolism data reported in this paper do not support a role for metabolic activation of DES in the mechanism of liver tumor formation in this animal model. Instead, it is conceivable that 7,8-BF and not the estrogen acts as the initiating compound in the hamster liver. There are two recent studies supporting this hypothesis: 7,8-BF is a potent clastogen and gives rise to DNA adducts in Chinese hamster ovary cells in the presence of rat liver microsomes induced with 2,3,7,8-tetrachloro-dibenzo~)dioxin (TCDD) [27]. Clastogenicity was not observed in the presence of PB-induced or uninduced microsomes. The genotoxicity of 7,8-BF in these studies has been attributed to its metabolic activation by specific P-450 isoenzymes which may be induced by TCDD but not by PB 1271. We have shown here and elsewhere [25,26] that pretreatment with 7,8-BF leads to 3-MC-like induction in the hamster liver. A recent study using the 32P-postIabeiling technique for the detection of DNA adducts has demonstrated that DNA adducts were only found in hamster livers after pretreatment with 7,8-BF alone or in combination with 17/I-estradiol but not with 17fi-estradiol alone nor in control animals [ZS]. This implies that 7,8-BF metabolites but not 17-estradiol metabolites are able to bind to DNA. In the hamster liver, neither DES nor 7,8-BF alone is able to induce hepatic tumors. By treating the hamster with DES plus 7,8-BF together, the 7,8-BF may act as the initiator and the estrogen DES as the promotor. Therefore, current experiments in our laboratory are aimed to investigate the metabolism of 7,8-BF in the hamster liver and its possible alterations by pretreatment with DES or 7,8-BF, and to identify the 7,8-BF metabolites capable of binding covalently to DNA. Acknowfedgemenw-This

tsche

study was supported by the DeuFor~hungsgemein~haft (Sonderfo~hungs~reich

172). We thank Mrs Hdla Raabe assistance and Mrs Jutta Colberg spectra. We are indebted to dear-Stiftung (Bamberg, F.R.G.) equipment.

for excellent technical for recording the mass the Doktor Robert for mass spectrometric

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