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Kidney International, Vol. 38 (1990), pp. 96—100

Oral shortens the prolonged bleeding time of uremic rats

CARLA ZOJA, GIANLuIGI VIGANO, DANIELA CORNA, MARIO SALMONA, GIUSEPPE REMUZZI, and SILvI0 GARATTINI

Mario Negri Institute for Pharmacological Research, 24100 Bergamo and 20157 Milano, Italy

Oral zeranol shortens the prolonged bleeding time of uremic rats. major side effects related to their hormonal activity, including Intravenous conjugated reduce the prolonged bleeding time in nausea, breast enlargement, a sense of congestion and heavi- uremic patients and in a rat model of uremia. However, estrogens have major side effects related to their hormonal activity. We investigated ness in the lower abdomen, hyperplasia of the endometrium and whether a f3-resorcylic acid lactone, zeranol (a compound with close endometrial carcinoma [15—17]. Thus, the search for molecules spatial similarity to estrogens but with a weak estrogenic activity),with a hemostatic activity comparable to improves primary hemostasis in uremic rats and whether the effect isbut devoid of side effects is of great importance to the future mediated by receptors. The results showed that single oral management of uremic patients with bleeding. In this context administration of zeranol significantly (P <0.01)shortened the bleeding time of uremic rats, 20 mg/kg being the minimum effective dose. This zeranol [18], a natural myco-estrogen with weak hormonal effect was long-lasting (72 hours). The dose of 30 mg/kg zeranolactivity [19], appears to be a potentially interesting candidate. reproduced the pattern observed after 20 mg/kg but bleeding timeZeranol (Fig. 1) is a $-resorcylic acid lactone employed as a values were still significantly (P <0.01)shortened 96 hours after thegrowth promoter in veterinary practice, and is remarkably less administration. No changes in hematocrit, platelet and leukocyte count, and serum creatinine were detected after zeranol administration. When active than as far as estrogenic potency [18]. Actually, uremic rats were pre-treated orally with two antago- Everett et al [20], comparing estrogenic potencies of resorcylic nists, and clomiphene (3 mg/kg), zeranol did not shorten the acid lactones and 17j3-estradiol in sexually immature female bleeding time, thus suggesting that the hemostatic effect of zeranol was rats, showed that the oral zeranol administration was 150 times due to an estrogen receptor-mediated mechanism. These results might less active in uterotrophic response than estradiol. An addi- have important future implications for the management of uremic bleeding in humans. tional important point is that this molecule is chemically unre- lated to but has a close tridimentional structure to estradiol, because it is of similar length and lipophylicity [18]. This remarkable spatial similarity explains the affinity of zera- Renal insufficiency is associated with a bleeding tendencynol for estrogen receptors [21—241. This permits a discrimination [1—4], due to a complex platelet dysfunction [1, 4—7], whichbetween affinity for estrogen binding sites and a steroidal-like correlates with a prolonged skin bleeding time [81. Modernstructure as the key determinant of the 'hemostatic' activity of techniques for the management of uremia have definitely re-a molecule. duced the incidence of severe bleeding episodes in renal failure Here we investigated whether zeranol given in a single oral patients, but hemorrhages still represent a major clinical prob-dose improved primary hemostasis in the rat model of chronic lem for patients undergoing surgery or invasive procedure [91.renal uremia, and whether its effect was inhibited by estrogen Several approaches have been proposed that shorten the pro-receptor blocking. longed bleeding time in uremia [reviewed in 9], among which conjugated estrogen infusions, by virtue of their long duration Methods of action, are of particular value when a long-lasting hemostatic 'competence' is required [10—121. Recently a rat model of Dose-effect study of zeranol on bleeding time in rats with chronic uremia has been established in which bleeding time is renal mass reduction prolonged and conjugated estrogens effective [13]. This model Male Sprague-Dawley rats (Charles River Italia S.p.A., establishes that the key component of the conjugated estrogenCalco, Italy), weighing 300 to 350 g at the start of the experi- mixture effective in shortening bleeding time is 17/3-estradiol,ment, were used. Animals were fed a standard rat chow and that its activity on primary hemostasis is mediated by an(Altromin Rieper, Vandoies, Italy) and had free access to tap estrogen receptor mechanism [14]. Estrogens, however, havewater. Renal mass was reduced by removing the right kidney and infarcting approximately five-sixths of the left kidney by ligation of two or three extrarenal branches of the main renal Received for publication July 31, 1989 artery, according to the method of Olson et al [25], with the rats and in revised form December 1, 1989 under ether anesthesia. Accepted for publication February 14, 1990 Four weeks after the ablative procedure 29 rats were subdi- © 1990 by the International Society of Nephrology vided as follows: group 1, eight rats which were given 10 mg/kg

96 Zoja et a!: Zeranol in uremic bleeding 97

HO 0 CH3 and interactions between anesthetics and drugs to be adminis- tered [261. Rats were placed iii a plastic cylinder with several openings from one of which the rat's tail emerged. A standard- ized Simplate II device (General Diagnostic, Milan, Italy) was used. The device was applied longitudinally on the dorsal part of the tail between 6 and 9 cm from the tip, taking care to avoid large veins. Tails of rats were left in the air and the animals were OH maintained at room temperature. Bleeding time was measured Fig. 1. Chemical structure of zeranol 16-(6,1O-dihydroxyundecyl)-f3- from the moment the tail was incised until bleeding stopped resorcyl acid lactone]. completely (no rebleeding within 30 seconds). Bleeding time was expressed in seconds (normal range: 80 to 120 seconds). zeranol orally (Compagnia di Ricerca Chimica, Udine, Italy) Laboratory tests dissolved in I ml of 0.5% carboxy methyl cellulose (Sigma Hematocrit values were determined by routine laboratory Chemical Co., St. Louis, Missouri, USA); group 2, eight ratsmethods. Platelets and leukocytes were counted by phase- which were given 20 mg/kg zeranol orally; group 3, eight ratscontrast microscopy. Serum creatinine was measured by the which were given 30 mg/kg zeranol orally; and group 4, five ratsalkaline picrate method [271. which received the vehicle alone. An additional group of 15 sham-operated rats (laparotomy and manipulation of renal Statistical analysis pedicles but without destruction of renal tissue) were divided in Results are expressed as mean SD. Statistical analysis was three groups (N =5)and given the different doses of zeranolperformed by using paired Student's (-test or one-way analysis orally. Bleeding times were measured before (time 0) and atof variance by Duncan test for multiple comparisons as appro- different time intervals after drug or vehicle administration. priate [28]. Statistical significance level was defined asP < 0.05.

Effect of zeranol on hematocrit, platelet and leukocyte count, Results and serum creatinine in rats with renal mass reduction Effect of zeranol administration on bleeding time in uremic To verify whether zeranol administration could affect hema- tological and renal function parameters, in six rats with renal rats mass reduction we measured hematocrit, platelet and leukocyte As previously reported [13] uremic rats had a significant (P < count, and serum creatinine before and 24, 48, 72 and 96 hours0.01) prolongation of bleeding time with respect to sham oper- after zeranol administration (20 mg/kg). The same parametersated rats [uremics (N =24)202 34 sec; sham (N =15)95 were measured in six sham-operated rats before and 24 hours16 sec]. after zeranol (20 mg/kg). Figure 2 shows the effect of different single doses of zeranol on bleeding time in uremic rats at different time intervals from Effect of estrogen receptor antagonists on bleeding time in drug administration. The dose of 10 mg/kg zeranol did not affect uremic rats treated with zeranol bleeding time as indicated by values recorded at 24 and 48 To evaluate whether zeranol's effect on bleeding time washours. At variance, 20 mg/kg zeranol significantly (P < 0.01) mediated by a receptor mechanism, we gave two estrogenshortened bleeding time. This effect lasted 72 hours. The receptor antagonists to uremic rats, namely tamoxifen andbleeding time values returned to normal range in five of eight clomiphene. These compounds are known to bind to cytoplas-rats at 24 hours, in seven of eight rats at 48 hours and in one of mic estrogen receptors, so that the modified complex is trans-eight rats at 72 hours. Ninety-six hours after zeranol adminis- located into the nucleus. A competition for estrogen bindingtration bleeding time values were again comparable to the basal sites results in a diminished amount of estrogen receptorones. Thirty mg/kg zeranol significantly (P < 0.01) reduced the available and explains their antiestrogenic activity [15]. bleeding time, and the effect lasted for 96 hours, with a Experiments were carried out in two groups of rats with renalsignificant (P < 0.01) shortening with respect to basal values. mass reduction (N =5each), receiving orally 3 mg/kg tamox-The bleeding time values returned to normal range in seven of ifen (Nolvadex, ICI-Pharma S.p.A., Milan, Italy) or 3 mg/kgeight rats at 24 hours, in all rats at 48 hours, in six of eight rats clomiphene (Clomid, Gruppo Lepetit, S.p.A., Milan, Italy) in 1at 72 hours and in five of eight rats at 96 hours. The adminis- ml of 0.5% carboxy methyl cellulose. The doses of tamoxifentration of vehicle alone had no effect on bleeding time in uremic and clomiphene were chosen taking into account the results ofrats (before vehicle: 19326; 24 hours after vehicle: 192 11 a previous study [14]. Bleeding times were measured before andsec). In sham operated animals different doses of zeranol had no 24 hours after estrogen receptor antagonists in order to evaluateeffect on bleeding time values (10 mg/kg: before: 95 21, 24 possible influences of estrogen receptor antagonist itself onhours after: 91 10; 20 mg/kg: before: 92 10, 24 hours after: bleeding time. Subsequently, zeranol (20 mg/kg) was orally85 11; 30 mg/kg: before: 98 16, 24 hours after: 99 9 sec). administered and 24 hours later bleeding time was again mea- sured. Effect of zeranol on hematocrit, platelet and leukocyte count and serum creatinine in uremic rats Bleeding time Twenty mg/kg, that is, the minimum dose which effectively Bleeding time was performed in unanesthetized animals toshortened the prolonged bleeding time of uremic rats, was used avoid possible influence of anesthesia on blood-vessel interplayto test whether zeranol affected hematological or renal function 98 Zoja et al: Zeranol in uremic bleeding

A 10mg/kg Table 1. Effect of zeranol administration (20 mg/kg) on hematocrit, 300 platelet and leukocyte count and serum creatinine in uremic rats ,Zeranol Hema- Platelet Leukocyte Serum 250 Time tocrit count count creatinine hours % 104/pJ 103/pJ mg/dl 0 43 2 88 6 14.9 3.5 1.04 0.17 24 42 3 105 27 15.6 4.5 1.09 0.18 48 41 2 100 7 13.8 5.9 1.07 0.17 72 13.9 5.1 1.12 0.18 100 40 3 87 5 96 41 1 88 9 11.6 2.8 1.09 0.14 50 Results are expressed as mean SD.

0 24 48 72 96 120

B 20 mg/kg Zerartol 300 300 ,ZeranoI 250] ,Tamoxifen 0 250 - 0200 E 150 -1 C, C V 200 0100-I 50J 150 - 0 24 48 72 96 120

C 30 mg/kg 0C 100 -j 3001 ZeranoI 0 250- E B Zeranol 0,300 200- C 0 0 1 ,Ctomiphene 150- 250 - 100- 50- 444A 0 24 48 72 96 120 200 - Time, hours Fig. 2. Effect of single oral zeranol administrations on bleeding time in uremic rats at different time intervals. A. 10 mg/kg. B. 20 mg/kg. C. 30 150 — mg/kg. Results are expressed as mean SD. *P< 0.01 as compared with basal bleeding time measurements. parameters in rats with renal mass reduction. As shown in 100- Table 1, no significant changes were observed in hematocrit 0 24 48 values, platelet and leukocyte counts, as well as in serum Time, hours creatinine measured before and at different time intervals afterFig. 3. Effect of estrogen receptor antagonists—tamoxifen (A) and zeranol. Similarly, in sham operated animals zeranol treatmentclomiphene (B)—on bleeding time in uremic rats receiving zeranol (20 mg/kg). Zeranol was given 24 hours after tamoxifen or clomiphene had no effect on hematocrit (before zeranol: 48 2 and at 24administration. Results are expressed as mean SD. hours: 49 2%), platelet count (before zeranol: 89 5 and at 24 hours: 88 4 x 104/1.d), leukocyte count (before zeranol: 11.9 1.9 and at 24 hours: 11.7 1.5 x 103/jsl) and serumSimilar results were obtained when the other estrogen receptor creatinine (before zeranol: 0.56 0.02 and at 24 hours: 0.58 antagonist, clomiphene, was used. These findings support the 0.04 mg/dl). possibility that an estrogen receptor-mediated mechanism is responsible for the hemostatic effectiveness of zeranol. Effect of estrogen receptor antagonists on bleeding time in uremic rats treated with zeranol Discussion As shown in Figure 3, the administration of 3 mg/kg tamox- The model of extensive renal mass ablation in the rat is of ifen blocked the zeranol effect on shortening the bleeding time.particular importance in studying the abnormal primary hemo- Zoja et a!: Zeranol in uremic bleeding 99

stasis associated with uremia, since bleeding time is prolongedestrogen-responsive cells induces a change in the receptor as it is in humans, and this abnormality is corrected bywhich promotes the translocations of the complex into the conjugated estrogens [13]. In the above rat model the effect ofnucleus with the subsequent induction of specific proteins [321. conjugated estrogens, given as a single injection, on bleedingSince like estradiol, zeranol induces the translocation of estro- time lasts for 48 hours (131, and is mainly attributable togen receptor sites to the nucleus [23, 33], one can postulate that 17/3-estradiol, one of the components of the mixture [14]. Inthe 'hemostatic' effect of zeranol is due to an identical mecha- patients with chronic renal failure, a long-lasting effect onnism or possibly to the induction of a different protein. Which is bleeding time was obtained after five infusions (0.6 mg/kg) ofthe receptor bearing cell that represents the target for the effect conjugated estrogens spaced 24 hours apart. The effect onof estrogen and zeranol on primary hemostasis is open to shortening the bleeding time lasted for 14 days or more,speculation. Actually, among cells involved in primary hemo- documenting a cumulative effect of repeated dosing of conju-stasis, possible candidates are endothelial or white blood cells gated estrogens [12]. that both possess estrogen receptors [34, 35]. Further studies With the present study we have documented that zeranol iswill properly clarify this important issue. also effective in shortening the prolonged bleeding time of In conclusion, we have documented that zeranol: 1) has a uremic rats. As for the estrogen mixture the effect of zeranol islong-lasting effect on the abnormal hemostasis of uremic rats; 2) long-lasting. Actually, a significant shortening of bleeding timeis active per os after a single administration; 3) its activity is was still observed 72 hours after 20 mg/kg and 96 hours after 30related to its binding to estrogen receptors. mg/kg zeranol administration. These results have obvious implications for the future man- Since estrogen treatment may be associated with potentialagement of uremic bleeding in humans. complications, particularly carcinoma and gynecomastia [15— 17], the present finding of the hemostatic effectiveness of Acknowledgments zeranol, a natural myco-estrogen with weak hormonal activity This study was partially supported by funds from the National [18, 20], seems to be of great importance. However, carefulInstitutes of Health (U.S.) grant no. HL 37491-03. The authors thank studies are required to exclude that a long-term zeranol treat-C.R.C. Compagnia di Ricerca Chimica S.p.A. (San Giovanni at Nati- ment may be associated with relevant side effects linked to thesone, Udine, Italy) for kindly providing zeranol, through the courtesy of Dr. Sergio Ferruzzi. The authors are also indebted to Dr. Magda estrogenic properties of the molecule. In a lifetime oral carci-Rossini and to Stefano Colombo for their cooperation. nogenicity study of zeranol in the rat [29] no evidence of any carcinogenic effect was reported, only mild endocrine-associ- Reprint requests to Carla Zoja, Biol.Sci.D., Mario Negri Institute for ated changes were observed in some organs. Endometrial Pharmacological Research, Via Gavazzeni, 11, 24100 Bergamo, Italy. changes have been described in long-term feeding studies of zeranol in the beagle dog and in the rhesus monkey [291. References However, no carcinogenic effect was observed in these animals 1. LEWIS JH, ZUCKER MB, FERGUSON JH: Bleeding tendency in after seven to ten years of zeranol administration. As far as uremia. 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