|Year : 2012 | Volume
| Issue : 6 | Page : 749-753
Gender differences in response to chronic treatment with 17β-oestradiol and 17β-aminoestrogen pentolame on hemostasis in rats
Cristina Lemini, Ruth Jaimez, Martha Medina-Jiménez, María Estela Ávila
Departament of Pharmacology, National Autonomous University of Mexico. Av. Universidad No. 3000, Ciudad Universitaria, Colonia Copilco Universidad, Delegación Coyoacán, Mexico City, Mexico
|Date of Submission||24-Apr-2011|
|Date of Decision||20-Feb-2012|
|Date of Acceptance||31-Aug-2012|
|Date of Web Publication||8-Nov-2012|
Departament of Pharmacology, National Autonomous University of Mexico. Av. Universidad No. 3000, Ciudad Universitaria, Colonia Copilco Universidad, Delegación Coyoacán, Mexico City
Source of Support: National Autonomous University of Mexico, Conflict of Interest: None
Objectives: This work evaluated chronic treatment with 17β-oestradiol (E 2 ) and 17β-aminoestrogen pentolame (AEP) on prothrombin time (PT), activated partial thromboplastin time (aPTT), thrombin time (TT), and fibrinogen concentration (FIB). Male (M) and ovariectomized (Ovx) Wistar rats were used to explore gender differences in the pharmacological response.
Materials and Methods: Rats (n = 12-18) were treated every third day during three months with E 2 (1, 10, 100 μg/kg), AEP (1, 10, 100, 500 μg/kg) or vehicle (propylenglycol 1 ml/ kg). PT, aPTT, TT, and FIB were measured using standardized techniques.
Results: Chronic treatment with E 2 in male rats increased PT (4-7%; P < 0.05), decreased aPTT (9%; 100 μg/kg; P < 0.05) and decreased TT (5% at 100 μg/Kg; P < 0.05). Chronic treatment with E 2 in ovariectomized female rats decreased PT (3-4%; P < 0.05), did not induce significant changes on aPTT and decreased TT in a dose dependent manner (12-27%; P < 0.05). Chronic treatment with AEP in male rats did not alter PT, increased aPTT in a dose dependent manner (5-16%; P < 0.05), and decreased TT (5%; 500 μg/Kg; P < 0.05) while in female ovariectomized rats it decreased PT (5-9%; P < 0.05), increased aPTT (8-13%; P < 0.05) and decreased TT (6-13%; P < 0.05). E 2 and AEP decreased FIB in M and Ovx animals. Decreases in FIB by E 2 were more pronounced in male (15-18% P < 0.05) than in ovariectomized rats (10-14% P < 0.05). E 2 showed more potency than AEP, lowering FIB at 1 and 10 μg/kg doses. Both estrogens decreased FIB in ovariectomized animals (E 2 , 10-14%, P < 0.05; AEP, 9% P < 0.05) and were reverted by increasing dosage.
Conclusions: Gender influenced response to chronic treatment with E 2 and AEP on hemostatic parameters. PT and aPTT were the most affected parameters, demonstrating non-equivalence in the pharmacological response of M and Ovx rats.
Keywords: 17β-aminoestrogens, gender, hemostasis, oestradiol, rat
|How to cite this article:|
Lemini C, Jaimez R, Medina-Jiménez M, Ávila ME. Gender differences in response to chronic treatment with 17β-oestradiol and 17β-aminoestrogen pentolame on hemostasis in rats. Indian J Pharmacol 2012;44:749-53
|How to cite this URL:|
Lemini C, Jaimez R, Medina-Jiménez M, Ávila ME. Gender differences in response to chronic treatment with 17β-oestradiol and 17β-aminoestrogen pentolame on hemostasis in rats. Indian J Pharmacol [serial online] 2012 [cited 2022 Oct 1];44:749-53. Available from: https://www.ijp-online.com/text.asp?2012/44/6/749/103287
| » Introduction|| |
Chronic oestrogen treatment is widely used in oral contraceptives (OC) and hormonal replacement therapy (HRT). One of the major concerns about its use is the possibility of producing thromboembolic events. Recent reports confirmed that users of currently available OC have over five times the risk of vein thrombosis compared to non-users and the risk can be higher during the first months of OC use.  The thrombotic effects of oestrogens have been associated with high doses, which produce a higher rate of thrombosis-related cardiovascular complications.  The usage of high oral doses even for a short period (i.e. emergency contraception) causes prompt effects on hepatic protein synthesis of coagulation factors, inducing significant alterations on blood coagulation.  Oral and transdermal contraception produce similar adverse effects on vascular risk markers. 
Hemostasis is a complex process that involves a balance of natural anticoagulants and procoagulant indicators.  In the formation of a thrombus vasoconstriction of vessels, platelet and blood coagulation activation occurs. A complex communication exists between endothelial cells, circulating platelets and the coagulation/fibrinolitic system. Physiologic estrogen levels seem to maintain a balance but supraphysiologic estrogen levels (or pharmacological doses) can alter the coagulation system, inducing an imbalance between anticoagulant and procoagulant factors that could lead to a prothrombic stage, predisposing to thrombosis risk. 
In the search of safe and efficacious new estrogensas therapeutic agents without thrombogenic risk, our group has been working on the biological activity characterization of 17β-aminoestrogens (AEs). These compounds in vivo and in vitro models produced estrogenic effects qualitatively similar to those of E 2 , but with a lower potency. , They decrease serum LH, increase uterine weight and behave similarly to E 2 as facilitators of sexual behavior in female rats. ,, Their oestrogenic actions are related to their capability of activating transcription through ER α and ER β receptors, preferentially binding to the ER α receptor. 
The AEs differ from E 2 in their ability to affect blood-clotting times (BCT) in rodents. Acute and sub-acute administration of high doses of E 2 or the17β-aminoestrogen pentolame (AEP) produce opposite effects on BCT in rats and mice. , E 2 shortens BCT, producing procoagulant effects, whereas AEP lengthens BCT producing anticoagulant effects.  The AEP and other 17β-aminoestrogens exert anti-platelet activity, reducing thrombi formation in mice and showing their potential as alternative non-thrombogenicestrogens. ,
Our experimental studies on the effects of oestrogens on blood coagulation have been mainly performed on male rats and mice. In some cases we have used ovariectomized (Ovx) animals, trying to eliminate the main source of oestrogens to avoid the female's cyclic hormonal activity. Previously we have reported gender intra and inter-species differences in some hemostatic coagulation markers in rats and mice (14). Ovariectomy in rodents is widely used to evaluate oestrogenic activity of any suspected estrogenic compound or to simulate clinical conditions such as menopause, where a decrease of oestrogen levels occurs. Ovariectomy itself provokes significant aPTT increases and FIB decreases compared with intact female rats. 
The use of male and Ovx rats in hemostasis studies is a common practice; however, few experimental reports have been published analyzing gender differences. On the other hand, it has been demonstrated that gender is an important factor in the variability of the pharmacological response. 
Considering the importance of this issue, the aim of this work was to study the effects of chronic treatment (CT) with E 2 and AEP [Figure 1] on males and Ovx Wistar rats using blood coagulation markers: prothrombin time (PT), activated partial thromboplastin time (aPTT), thrombin time (TT), and fibrinogen concentrations (FIB).
|Figure 1: Chemical Structures of oestradiol (E2) and the 17β-aminoestrogen pentolame (AEP)|
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| » Materials and Methods|| |
17β-estradiol (E 2 ; 1, 3, 5(10)-estratrien-3,17-beta-diol) was obtained from Syntex (Mexico). Pentolame (AEP; 17β-(5-hydroxy-1-pentylamino)-1, 3, 5(10)-estratrien-3-ol) [Figure 1], was synthesized from estrone: 3-hydroxy-1, 3, 5(10)-estratrien-17-one (Syntex, Mexico). Chemical purity of pentolame was established comparing its spectral (IR, NMR, MS) and chromatographic (HPLC and TLC) properties with that previously reported.  All chemicals used were of the highest purity available from Baker Co. (Mexico).
All experimental studies were conducted in accordance to the Mexican National Protection Laws of Animal Welfare (NOM-062-Z00-1999) and were bred in the animal housing facilities of the School of Medicine, UNAM. Adult Wistar male and female rats weighing 200 to 250g were used. Female Wistar rats were ovariectomized under chloral hydrate anesthesia before treatment. The animals were kept at a constant temperature (20-22°C) in a room with 12:12 h light-dark cycle and maintained on standard chow (Nutricubos, Purina) and water ad libitum .
Experimental Design of Chronic Treatment
Twenty-one days after ovariectomy rats were distributed among groups according to the balanced Latin-square block design according to their body weight (5-6 animals per group in each experiment). Different groups of male and Ovx rats were administered with E 2 (1, 10, 100 μg/kg), AEP (1, 10, 100, 500 μg/kg) or vehicle (propyleneglycol 1 ml/kg) for three months every third day. The experiment was carried out twice.
Blood collection: Animals were anesthetized with chloral hydrate (4% solution, 7 ml/kg) prior to blood withdrawal. Each animal was placed on its back on a cork surgery table and restrained with string fixed at the corners. Arterial blood was collected from the iliac bifurcation with a plastic syringe and disposable-gauge needles (21 × 32 mm), which provided hemolysis-free blood samples (4-7 ml). Blood was immediately drawn out into plastic tubes containing 0.11 M sodium citrate (1:9, v:v). The samples were gently mixed and centrifuged at 2500 g for 10 minutes at 4°C. Plasma samples were separated and stored at-70°C until processing.
Hemostatic parameters: The coagulation screening tests, prothrombin time (PT), activated partial thromboplastin time (aPTT), thrombin time (TT), and fibrinogen concentrations (FIB), were performed with modifications of the conventional clinical procedures described earlier.  For all clotting tests, Dade® Behring reagents were used and assayed according to instructions in the package insert. Reference curves and accuracy controls were set up using the corresponding control plasma of the manufacturer. PT, aPTT, and TT clotting times were recorded using a Behring Fibrintimer II (Dade® Behring). Clot formation was the end point of the reactions and results are reported in seconds. PT determination was obtained using rabbit brain thromboplastin C plus (Dade® Behring). Plasma samples (50 μl) were pipetted into cuvettes and then 100 μl of thromboplastin C plus were added to activate the reaction. aPTT was assessed using the Actin FS reagent (Dade® Behring) containing purified soy phosphatides. Actin FS (100 μl) was added to plasma samples (50 μl). The mixture was incubated at 37°C for 120 s and 50 μl of 0.02 M CaCl 2 (37°C) was added to activate the reaction. TT was determined using bovine thrombin (Dade® Behring) at a concentration of 5 IU/ml. Plasma samples (50 μl) were treated with 50 μl of bovine thrombin to start the reaction. FIB concentration was evaluated in a mechanical fibrometer Fibrosystem Becton-Dickinson Mod 5-117V. Bovine thrombin (100 IU/ml; Dade® Behring) was added to plasma samples (50 μl) to induce clot formation. The FIB concentration was obtained from a reference curve calibrated with human plasma fibrinogen and reported in milligrams per deciliter (mg/dl).
Statistical significance between a control group that received vehicle and the treated groups was assessed by analysis of variance (ANOVA). The statistical analysis was performed using the Sigma Stat 3.1 software. The significance of differences among groups was estimated by the Dunn's or Tukey's test as appropriate. Results were expressed in means ± standard error (S.E.M.). P < 0.05 values were considered as the limit of significance. The differences related to the controls for all the hemostatic parameters were obtained by the relation: [(Mean value treated group/Mean value control group) × 100] -100. All experiments were performed twice and the samples of the evaluated parameters were assessed in triplicate (n = 12-18).
| » Results|| |
Chronic treatment (CT) of male and Ovx Wistar rats was performed every third day along three months with either E 2 (1, 10, 100 μg/kg), AEP (1, 10, 100, 500 μg/kg), or vehicle (propyleneglycol 1 ml/kg) and the effects on their hemostatic parameters: PT, aPTT, TT, and FIB are shown in [Figure 2].
|Figure 2: Effects of chronic administration of E2 (1, 10, 100 μg/kg) and AEP (1, 10, 100 and 500 μg/kg) on PT, aPTT, TT, and FIB in male and Ovx Wistar rats. *P < 0.05 vs. vehicle. Each point represents the mean ± S.E.M of 15-18 samples|
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E 2 administration in males increased PT (7, 7, and 4%; P < 0.05) and no significant changes were seen after AEP administration, whereas E 2 and AEP in Ovx decreased PT (E 2 ; 4%, 3%, 4%; P < 0.05; AEP; 8%, 9%, 7%, 5%; P < 0.05).
In male rats, E 2 decreased aPTT (9%; 100μg/kg; P < 0.05) and AEP increased it in a dose dependent manner (5, 9, 16%; 10, 100, 500 μg/kg; P < 0.05). In Ovx animals, E 2 did not induce significant changes on aPTT, whereas AEP increased aPTT significantly with the three highest doses (13%, 8%, 13%; P < 0.05 respectively). E 2 and AEP decreased TT in both male and Ovx. In males they decreased TT~5% at their highest doses (P < 0.05); In the Ovx rats, both steroids decreased TT in a dose dependent manner, E 2 being more potent than AEP (E 2 12, 23, 27%; AEP 6, 7, 12 and 13%; P < 0.05). E 2 and AEP decreased FIB in both groups. On male rats the decrease in FIB was more pronounced (E 2 15, 18 and 16%; AEP 10, 10, 14, 11%; P < 0.05) than that elicite d on Ovx animals (E 2 ; 14, 10, (P < 0.05); 3% (NS); AEP, 9%; (P < 0.05), 5, 3, 2% (NS). E 2 also showed more potency than AEP. In Ovx animals FIB decreased with both estrogens at their lowest doses however the effect was reverted by increasing the dosage.
| » Discussion|| |
Our results showed that CT of male and Ovx rats with E 2 and AEP produced differential actions on blood coagulation markers.
Male rats chronically treated with E 2 showed significant alterations of PT, aPTT, TT and FIB, showing the compound's influence on the intrinsic, extrinsic and common pathway of coagulation. aPTT and TT were less affected since only the highest doses induced significant but modest decreases in both parameters. In contrast, AEP in males did not modify PT at any assayed dose and induced opposite responses to those of E 2 on aPTT. E 2 showed a tendency to decrease aPTT while AEP produced a dose dependent increase of this parameter.
In Ovx both oestrogens decreased PT however, AEP decreased PT values twice as much as E 2 at 10 μg/kg. This effect is opposite to that observed in male rats relative to its control values. The response of AEP was similar in both males and Ovx however, in Ovx the response was not dose dependent. E 2 produced a more pronounced decrease of TT and FIB values compared to AEP suggesting that gender has an important influence in the response to oestrogens in blood coagulation markers.
The decreasing responses of FIB after treatment with E 2 and AEP were more pronounced in males than in Ovx rats. We found basal FIB levels significantly lower (~25%) in Ovx vs males. This data is in agreement with that reported by Oyekan and Botting  and Emmes et al., , however, the latter found that FIB values in males doubled those of female Wistar rats. The differences in our results could probably be explained by the hormonal status of the intact female animals, considering that our experiments used Ovx animals.
Gender differences have been observed in the hemostatic system of rats. Male and intact female rats have showed differences in thrombus formation related to oestrogen levels and have an influence on coagulation markers and platelet reactivity. , The highest thrombus formation is in the diestrus stage of the estrous cycle of female rats when estrogen levels are low. Thrombus deposition is higher in males compared to female rats,  and E 2 administration to both male and female rats reduced thrombus formation.  On the other hand, rat gender differences in intravascular aggregation of platelets and the fibrinolytic pathway have been found.  Male clot lysis time is longer and the fibrinogen levels and plasminogen activity is higher than in female rats. 
A great number of clinical and experimental studies under physiological and pathological conditions have found gender related differences in the cardiovascular system.  It is also well documented that gender differences play an important role in hemostasis and vascular reactivity.  The therapeutic implications of the gender-specific aspects of cardiovascular disease have been revised. Gender-specific differences have been recognized to influence the pharmacological response (15). Clinical reports have also revealed gender response differences to pharmacological medications used to prevent and treat cardiovascular disease.  Sex differences in drug responses have been related to genetic variations, hormonal status and differences in the pharmacokinetic and/or pharmacodynamic processes. 
Effects of E 2 and AEP administered to rats probably include genomic action mechanisms involving the activation of specific receptors that interact with co-regulatory proteins, which could stimulate or inhibit target transcription genes. Other pathways can be activated or inhibited since steroids also exert their effects acting at the membrane site level in a non-genomic mode, promoting or inhibiting other specific path signals.  The response observed probably involved the steroids action on genomic and non genomic events over the complexity of the hemostatic system. The mechanism involved in the anticoagulant and anti-thrombic effect of the AEP probably implicates changes in the activation of the coagulation system and could also be due to its effects induced at the membrane site. It has been recently described that prolame a close structurally related AEP analogue induces NO production in endothelial cells that affects responsive platelets aggregation events where a non-genomic mechanism could be implicated. ,
In the thrombus formation vasoconstriction of the vessels, platelet and blood coagulation activation are involved. A complex communication exists between endothelial cells and circulating platelets and the coagulation/fibrinolitic system (5). It has been demonstrated that the natural hormone 17β-oestradiol (E 2 ) modulates the transcription of some blood coagulation gene factors (FVII and FXII).  The human genome contains high affinity estrogen response elements (EREs) in genes belonging to the procoagulant and anticoagulant pathways (II, V VII, IX, X, XI, XII, protein S, protein Z and heparin cofactor II). , Hemostasis is a complex process that involves a balance of natural anticoagulants and procoagulant indicators.  Physiologic oestrogen levels seem to maintain the balance but supraphysiologic oestrogen levels (or pharmacological doses) can alter the coagulation system, inducing an imbalance between anticoagulant and procoagulant factors that could lead to a prothrombic stage, that predispose to the thrombosis risk. 
Our results showed response differences between the two assayed oestrogens within the same gender group and allowed us to infer that different pathways of the blood coagulation cascade could be affected by the dose and oestrogens nature on the same gender. CT of male and Ovx rats with E 2 and AEP did not induce equivalent pharmacological responses in the blood coagulation system; rather they induced different effect profiles. Ovx animals used to study oestrogen responses on blood coagulation is a useful tool to avoid endogen oestrogens production however, Ovx and male animals are not equivalent in the hemostasis response.
This work suggests the need for further experimental studies focused on finding a gender-specific drug assessment. Drugs research must take into account gender response differences to allow a proper comparison between the pharmacological effects of oestrogens and hemostasis.
| » Acknowledgments|| |
This study was supported by grant from National Autonomous University of Mexico, DGAPA IN211908-2.
| » References|| |
|1.||Van Hylckama Vlieg A, Helmerhorst FM, Vandenbroucke JP, Doggen CJ, Rosendaal FR. The venous thrombotic risk of oral contraceptives, effects of oestrogen dose and progestogen type: Results of the MEGA case-control study. BMJ 2009;339:b2921. |
|2.||Gembitskii EV, Begunov AB. Changes in parameters of the coagulation and fibrinolysis system of the blood in patients with prostate cancer in the framework of estrogen therapy. Klin Med (MosK) 1994;72:30-2. |
|3.||Van Rooijen M, Silveira A, Thomassen S, Hansson LO, Rosing J, Hamsten A, et al. Rapid activation of haemostasis after hormonal emergency contraception. Thromb Haemost 2007;97:15-20. |
|4.||Vehkavaara S, Silveira A, Hakala-Ala-Pietilä T, Virkamäki A, Hovatta O, Hamsten A, et al. Effects of oral and transdermal estrogen replacement therapy on markers of coagulation, fibrinolysis, inflammation and serum lipids and lipoproteins in postmenopausal women. Thromb Haemost 2001;85:619-25. |
|5.||Furie B, Furie BC. The molecular basis of blood coagulation. Cell 1988;53:505-18. |
|6.||Van Hylckama Vlieg A, van der Linden IK, Bertina RM, Rosendaal FR. High levels of factor IX increase the risk of venous thrombosis. Blood 2000;95:3678-82. |
|7.||Lemus AE, Jaimez R, Lemini C, Menjivar M, Silva G, Rubio-Póo C, et al. Estrogenic effects of the synthetic aminoestrogen 17β-(5-hydroxy-1-pentylamino)-1,3,5(10)-estratrien-3-ol (pentolame). Steroids 1998;63:433-8. |
|8.||Jaimez R, Coney A, Jackson K, Lemus AE, Lemini C, Cárdenas M, et al. In vivo estrogen bioactivities and in vitro estrogen receptor binding and transcriptional activities of anticoagulant synthetic 17β-aminoestrogens. J Steroid Biochem Mol Biol 2000;73:59-66. |
|9.||Lemini C, Canchola E. Effects of 17β-aminoestrogens on the sexual behavior of female rats. Physiol Behav 2009;96:662-6. |
|10.||Lemini C, Rubio-Poó C, Silva G, García-Mondragón J, Zavala E, Mendoza-Patiño N, et al. Anticoagulant and estrogenic effects of two new 17β-aminoestrogens, butolame [17β-(4-hydroxy-1-butylamino)-1,3,5(10)-estratrien-3-ol] and pentolame [17β-(5-hydroxy-1-pentylamino)-1,3,5(10)-estratrien-3-ol]. Steroids 1993;58:457-61. |
|11.||Lemini C, Franco Y, Avila ME, Jaimez R. Contrasting effects of estradiol and 17 beta-aminoestrogens on blood clotting time in rats and mice. Eur J Pharmacol 2005;510:229-33. |
|12.||De la peña A, Baños G, Izaguirre R, Tobòn-Cervantes A, Fernàndez-G JM.Comparative effect of three synthetic alkanol-amino-estrogens; Etolame, buolame and pentolame, on platelet aggregation. Med Sci Res 1994;22:119-20. |
|13.||González G, Alvarado-Vasquez N, Fernández-GJM, Cruz-Robles D, Del Valle L, Pinzón E, et al. The antithrombotic effect of the aminoestrogenprolame (N-(3-hydroxy-1,3,5(10)-estratrien-17B-YL)-3-hydroxypropylamine) is linked to an increase in nitric oxide production by platelets and endothelial cells. Atherosclerosis 2009;208:62-8. |
|14.||Lemini C, Jaimez R, Franco Y. Gender and inter-species influence on coagulation tests of rats and mice. Thromb Res 2007;120:415-9. |
|15.||Franconi F, Brunelleschi S, Steardo L, Cuomo V. Gender differences in drug responses. Pharmacol Res 2007;55:81-95. |
|16.||Oyekan AO, Botting JH. Relationship between gender differences in intravascular aggregation of platelets and fibrinolytic pathway in the rat. Arch Int Pharmacodyn Ther 1991;313:176-92. |
|17.||Emms H, Lewis GP. Sex and hormonal influences on platelet sensitivity and coagulation in the rat. Br J Pharmacol 1985;86:557-63. |
|18.||Emms H, Lewis GP. The effect of ovarian hormones on an in vivo model of thrombosis in the rat. Br J Pharmacol 1985;84:243-8. |
|19.||Ostadal B, Netuka I, Maly J, BesikJ, Ostadalova I. Gender differences in cardiac ischemic injury and protection experimental aspects. Exp Biol Med (Maywood) 2009;234:1011-9. |
|20.||Schwertz DW, Penckofer S. Sex differences and the effects of sex hormones on hemostasis and vascular reactivity. Heart Lung 2001;30:401-26. |
|21.||Regitz-Zagrosek V. Therapeutic implications of the gender-specific aspects of cardiovascular disease. Nat Rev Drug Discov 2006;5:425-39. |
|22.||Ellmann S, Sticht H, Thiel F, Beckmann W, Strick R, Strissel PL. Estrogen and progesterone receptors: From molecular structures to clinical targets. Cell Mol Life Sci 2009;66:2405-26. |
|23.||Citarella F, Misti S, Felici A, Aiuti A, Laporta C, Fantoni A. The 5¢ sequence of human factor XII gen contains transcription regulatory elements typical of liver specific, estrogen-modulated genes. Biochim Biophys Acta1993;1172:197-9. |
|24.||Di Bitondo R, Hall AJ, Peake IR, Iacoviello L, Winship PR. Oestrogenic repression of human coagulation factor VII expression mediated through an oestrogen response element sequence motif in the promoter region. Hum Mol Genet 2002;11:723-31. |
|25.||Bourdeau V, Deschenes J, Metivier R, Nagai Y, Nguyen D, Bretschneider N, et al. Genome-wide identification of high-affinity estrogen response elements in human and mouse. Mol Endocrinol 2004;18:1411-27. |
[Figure 1], [Figure 2]