Europ. 07. Cancer Vol. 5, pp. 509-513. Pergamon Press 1969. Printed in Great Britain

The Formation of Diethylstilbestrol Monoglucuronide in Hamster Liver Homogenates

MARIA GABALD()N and TERESA LACOMBA Servicio de Cancerologia Experimental, Faeultad de Medicina,

Paseo de Valencia al Mar, Valencia, Spain

I N T R O D U C T I O N VAsQuv.z-LoPEz [1] observed for the first time the presence of renal tumours in the male golden hamster treated with diethylstilbestrol (DES). Later studies confirmed the appearance of renal tumours in male [2-5] and ovariect- omized female hamsters [6] but not in other animals of experimentation after administra- tion of natural estrogens or DES. These tumours are hormone-dependent and trans- plantable [7]; in the first transfers they keep their hormonal dependency but after successive generations become developed without external estrogen contribution [8, 9].

This phenomenon is of great interest as it is a hormone-induced tumour in an organ which is not normally regarded as belonging to the endocrine system. Horning [10] has formu- lated the hypothesis that hamster liver had a smaller DES conjugation capacity than other rodents; this fact would cause non-conjugated high DES levels to be eliminated becoming an irritative stimulus to the hamster kidney, eventually leading to tumour formation.

In view of Horning's hypothesis and the little known about DES metabolism we thought it worthwhile to do a comparative study of UDP glucuronyl transferase activity with DES as substrate in rat and hamster livers. Due to the fact that estrogens cause a decrease in liver glucuronyl transferase activity [11], we have considered of interest to also study the in vivo effect of DES upon hamster liver.

Submitted for publication 21 April 1969. Accepted 18 June 1969.

509

MATERIAL AND METHODS Chemicals. DES, uridin diphosphate glucur-

onic acid (UDPGA) and saccharic acid were provided by Sigma Chemical Co. ; tris(hydroxy- methyl)aminomethane "Tris", propylene gly- col and perchloric acid, by Schuchardt; [3-glucuronidase (Ketodase) 5000 units/ml by Warner Chilcott and 25 mg DES pellets (Cyren A), by Bayer. The other reagents were of ana- lytical grade, the solvents were redistilled and diethyl ether was maintained free from perox- ides by storage over ferrous sulfate and was freshly distilled before use. Saccharolactone approximately 0.016 ~ was obtained starting from a 0.05 M saccharic acid solution following Dutton's method [12]. Diethylstilbestrol mono- glucuronide (DESGA) was obtained from the urine of rabbits fed DES at a daily rate of 500 mg for 5 days following the procedures given by Dodgson et al. [13].

Animals. The following types of animals were used: (1) three months old male hamsters; (2) three months old female hamsters; (3) eleven months old male hamsters, castrated at three months; (4) hamsters of types 1, 2 and 3 seven days after a 25 mg DES subcutaneous pellet implantation; (5) three months old male Wistar rats.

The animals were sacrificed by cervical dislocation and the livers immediately removed and washed with 0.15 u KCI-3-2×10 -4 M KHCO8 solution at 0°C.

Determination of UDP glucuronyl transferase activity. It has been done by measuring the DES consumed after an incubation period with the liver homogenate, following the technique

510 Maria Gabalddn and Teresa Lucomba

Table. 1. DES consumption by hamster and rat liver homogenates*7

Without UDPGA With UDPGA

Castrated $ hamster [lO]$ -0.2f7.5 64.4-j--9.2

Castrated and implanted 3 hamster [lo] 2.156.7 74.4h9.3

3 hamster [lo] 9.7f 18.2 77.6f8.2

Implanted $ hamster [8] 2.1&6.3 64.7& 16.6

9 hamster [9] 15.06 10.0 67.5k13.8

Implanted 9 hamster [I I] 6.3h7.4 50.8& 15.7

3 rat [lo] 3.1f5.7 12.3&8.6

*The composition of the incubation mixture was as follows: tris, 35 mM; MgClse6Hs0, 10.5 mM; UDPGA, 0.67 mM; DES, 0.149 mM; 3% v/v propylene glycol; 15%homo- genate, 1 ml; final volume, 5 ml; pH, 7.4.

tDE.3 consumption is expressed as mcg/l50 mg of fresh liver/90 min at 37°C. Results are given as the mean & S.D.

3Number-s in parentheses indicate number of animals used.

described by Gabaldon et al. [ 141. The amount of DES consumed was calculated by difference with a blank incubated without substrate and with DES added after protein precipitation. Assays were done in triplicate. The compos- ition of the incubation mixture is described in Table 1.

DESGA identijication. The volume and time of incubation used to identify DESGA, were twice that of the quantitative assays. The reaction was stopped with 2 ml of 12 - 5% perchloric acid and 20 min later, 8 ml of ethanol was added. After 20 min the mixture was filtered; 0.5g of KHCOs was added to the filtrate in order to eliminate excess HC104, KC104 being separated by cold filtration. The filtrate was extracted four times with an equal volume of diethyl ether. From here on, methods given by Gabaldon et al. [14] were followed. The final residue obtained was dissolved in 0 * 5 ml of 80% ethanol. An aliquot was applied to two silicagel plates; one plate was developed in ethyl acetate:2- propanol: water (6:2*5 :l -5) and the other in n-butanol : acetic acid : water (6 :2 :2) ; both plates were sprayed with 50% H&O4 and heated to 100°C for 3 min. Another aliquot was subjected to hydrolysis with p-glucuronidase. The com- position of the incubation mixture was as follows : extract, 0 - 15 ml; P-glucuronidase, 1500 units ; saccharolactone, approximately O-5 mM; acetate buffer pH 4.5, 85 mM; final volume, 3 ml. It was incubated at 37°C for 90 min, evaporated to dryness and the residue dissolved in O-5 ml of 80% ethanol. An aliquot was applied to a silicagel plate, devel- oped in benzene: ethanol (9 :l) and DES detected by spraying with O-5 N Folin-Cio- calteu’s reagent and exposure to ammonia vapor.

RESULTS Table 1 shows DES consumption in absence

and presence of UDPGA by liver homogenates of male rat, male, castrated male and female hamsters and by liver homogenates of the same type of hamsters a week after a 25 mg DES pellet subcutaneous implantation. DES con- sumption is expressed in mcg/150 mg of fresh liver/90 min at 37°C. In absence of UDPGA, DES consumption is negligible and variable and can be due to residual UDPGA levels or to other unknown pathways of utilization.

The statistical significance levels of differ- ences in DES consumption by rat and hamster liver homogenates have been determined. Each one of the variables has been studied : sex, DES pellet implantation, castration and species. Statistical significance levels have been calcu- lated according to the Student t-test. Differ- ences are considered significant when P<O * 0 1.

DES consumption differences in presence and absence of UDPGA are highly significant in all cases. In absence of UDPGA none of the variables studied determines statistically signi- ficant differences of DES consumption. In the presence of UDPGA, only species and castra- tion determine values of P<O * 01. In this last case considering the differences of age of castrated animals with respect to the intact, differences found could be due to age instead of castration.

When the ethanolic extract obtained from the incubation mixture (see experimental) was subjected to chromatography development in ethyl acetate : 2-propanol : water (6 :2 * 5 : 1.5) and n-butanol: acetic acid: water (6:2:2), a spot appeared having the same RI (0 * 3 and 0 - 82 respectively) as standard DESGA ob- tained from rabbits urine treated with DES. The incubation of the ethanolic extract with

The Formation of Diethylstilbestrol Monogluatronide in Hamster Liver Homogenates 511

[~-glucuronidase liberated DES which was detected by thin layer chromatography. DES formation was inhibited in presence ofsaccharo- lactone. DESGA has not been detected in ethanolic extracts obtained from a DES-free incubated mixture. Detection of DESGA has been accomplished with positive results in all series, being the only metabolite detected under conditions utilized.

DISCUSSION In liver of animals treated with DES a

decrease of glucuronyl transferase activity could be expected due to an estrogen depressor effect upon that activity. Nevertheless in implanted animals no differences have been observed in DES consumption in relation to non-treated animals even if the estrogen is absorbed during this period as shown by the decrease in pellet weight observed seven days after implantation.

Liver UDP-glucuronyl transferase levels are usually higher in males than females [15]. Even so, it has been known that in hamster the conjugation capacity of progesterone with glucuronic acid is higher in female than in male. In our case sex has not determined statistically significant differences of DES consumption neither in control nor in implan- ted animals.

Horning suggested that the renal tumour in hamster produced by DES could be due to a diminished liver conjugation capacity as com- pared to other rodents that do not exhibit the phenomenon. Later reports by Fishman and Sie [16] and Rao and Taylor [17] sustained this point of view by showing that the production of glucuronides from testosterone and proges- terone was lower in hamster liver than in rat liver and at the same time smaller in male hamster than in female.

I fHorning 's hypothesis were true, glucuronyl transferase activity with DES as substrate should be higher in rat than in hamster and

in this last animal greater activity in female than in male. Our results do not agree with this hypothesis as we have found activity in hamster to be from 5 to 6 times higher than in rat and the differences between the sexes have not been statistically significant.

It must be taken into consideration that in our experiences the UDPGA concentration does not constitute a limiting factor, while in vivo the levels of the same can be decisive for the conjugation reaction. Wong and Sourkes [18] found that liver UDPG levels are considerably lower in hamster as compared to other experimental animals, the hamster level being one third that of rat. UDPGA level is identical in both animals. This attainability deficiencies for UDPG could, in heavy require- ment cases, determine an in vivo precarious conjugation reaction.

Our results obtained in vitro together with Kirkman's experiences [8] who failed in the at tempt to produce renal tumours by DES pellet implantation in the spleen or in the path of the hepatic portal drainage suggest that the liver is not involved directly in the renal tumour phenomenon produced by DES in hamster.

Ghaleb [19] while working with labelled DES found that the tracer hormone was taken up and bound to renal cellular proteins, being the amount of labelled DES concentrated in renal epithelium double in male hamster than in female. In view of this it seems more reasonable to explain the mechanism of renal tumour by DES as a hormone balance alteration in which cellular receptors would have a predominant role.

Admowledgements---This investigation was per- formed under the auspices of the Asociaci6n Espafiola contra el Cancer and was supported in part by the help of Pfizer Laboratories and by grant given to Dr. Gabald6n by the Comisarla de Protecci6n Escolar del Ministerio de Educaci6n y Ciencia.

RESUME On a d/montrd, in vitro, la rgaction de conjugaison du di/thylstilboestrol (DES) avec l'acide glycuronique sur des homoggnats de foie du hamster dor/ et du rat. La mesure de l'activit6 de UDP-glycuronyl transfgrase a dt/ r/alisge par la dgtermination photocolori- m/trique du DES consommg, apr~s une pgriode d'incubation avec l'homogdnat. Le digthylstilboestrol monoglycuronide ( DESGA) a/ tg identifi/ comme un produit de rgaction par la chromatographie sur couche mince, et par r hydrolyse enzymatique par la ~-glycuro- nidase. On a fai t une gtude comparative des activit/s de l' UDP-glycuronyl transfdrase chez le rat mdle, le hamster mdle, le mdle castr/ et la femelle, et sur les mgmes groupes d' animaux, une semaine apr~s l'implantation sous-cutan/e d'une pilule de 25 mg de DES.

En l'absence de UDPGA, il n'y a pour ainsi dire aucune consommation de DES clans aucun des cas. Des variables gtudiges, sexe, castration, implantation et esp&e, seule

512 Mar&i Gabaldon and Teresa Lacomba

cette de&&e d&ermine des d@rences statistiquement signa$catives de consommation de DES. Le foie du hamster consomme une quantitd de DES six fois supt%eure ci celui du rat. On discute le r61e du foie darts le processus de la genese de la tumeur du rein chet le hamster par le DES.

SUMMARY The in vitro DES conjugation reaction with glucuronic acid in golden hamster and rat liver homogenates has been made evident. The measurement of UDP-glucuronyl trans-

ferase activity has been done by photocolorimetric determination of DES consumed after an incubation period with the homogenate.

DESGA has been chromatographically and enzymatically ident$ed as a reaction product. A comparative study of activity in male rat, male, castrated male and female hamsters and in the same type of hamsters a week after a 25 mg DES pellet implantation, has been done.

In absence of UDPGA there is practically no DES consumption in any one of the cases. Of all variables under study: sex, castration, implantation and species, on& this last one determines DES consumption di$erences of high statistical sign$cance. Hamster liver consumes six times as much DES as that consumed by rat liver. The role of liver in renal tumour processes in hamster by DES is discussed.

ZUSAMMENFASSUNG Es wurde in vitro die Konjugation von Diaethylstilboestrol (DAS) mit Glukuronsaure in Leberhomogenaten von Goldhamstern und Ratten nachgewiesen. Die UDP-Glucuronyl- transferaseaktiviti wurde durch photocolorimetrische Messung des nach einer Inkubations- zeit mit dem Homogenat verbrauchten Diaethystilboestrols bestimmt. DASG ist chroma- tographisch und enzymatisch als Reaktionsprodukt identa&iert worden. Es wurde vergleichend eine Aktivitiitsbestimmung bei m&u.4chen Ratten, miinnlichen kastrierten Ratten und weiblichen Hamstern, sowie im gleichen Hamstertyp, eine Woche nach der Implantation von Pillen aus 25 mg DAS, durchgeftihrt. In Abwesenheit von UDPGA besteht praktisch in keinem Fall ein DAS- Verbrauch. Von allen experimentellen Variablen wie Geschlecht, K&ration, Implantation und Species hat nur die letztere Eintuj auf Unterschiede im DAS-Verbrauch, die von hoher statistischer Signijikanz sind. Die Leber des Hamsters verbraucht sechsmal soviel DAS wie die Leber der Ratte. Die Rolle der Leber bei renalen Tumorentwicklungen im Hamster durch DAS wird diskutiert.

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The Formation of Diethylstilbestrol Monoglucuronide in Hamster Liver Homogenates 513

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