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Gynecol Endocrinol, Early Online: 15
! 2014 Informa UK Ltd. DOI: 10.3109/09513590.2014.943727

ORIGINAL ARTICLE

Antioxidant effect of the active metabolites of tibolone

Julia Stark1*, Szabolcs Varbiro2*, Miklos Sipos2, Zsolt Tulassay1, Levente Sara2, Ildiko Adler1, Elek Dinya3,
Zoltan Magyar4, Bela Szekacs1, Istvan Marczell1, Helenius J. Kloosterboer5, Karoly Racz1, and Gabor Bekesi1

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2nd Department of Internal Medicine, Faculty of Medicine, 22nd Department of Obstetrics and Gynecology, Faculty of Medicine, Semmelweis
University, Budapest, Hungary, 3Faculty of Health and Public Services, Institute of Health Informatics Development and Further Training,
Semmelweis University, Budapest, Hungary, 41st Department of Obstetrics and Gynecology, Faculty of Medicine, Semmelweis University, Budapest,
Hungary, and 5KC2, Oss, The Netherlands
Abstract

Keywords

Certain steroidal compounds have an antioxidant effect in humans. Our aim was to test
whether the synthetic steroid tibolone and its metabolites are also able to display such a
property. For this, granulocytes from healthy men and women were incubated for two
hours with different concentrations (10 7, 10 8, 10 9 M) of either estradiol, tibolone, 3ahydroxytibolone, 3b-hydroxytibolone, D4-tibolone, 3a-sulfated-tibolone, 3a-17b-disulfatedtibolone, 3b-sulfated-tibolone or 3b-17b-disulfated-tibolone. Superoxide anion generation of
neutrophils was measured by photometry. Results of different steroids were given as
percentages of their controls. A more simple superoxide generating system, the xanthine
xanthine oxidase reaction was also tested. We found that granulocyte superoxide production
did not differ from the control using 10 9 M of steroids. Using 10 8 M concentration: estradiol
(80.9 2.5%); 3b-sulfated-tibolone (83.3 4.7%); 3b-17b-disulfated-tibolone (81.0 4.2%)
caused a significant decrease in superoxide production, compared to the control. In addition
at 10 7 M, 3b-hydroxytibolone and 3a-sulfated-tibolone also showed antioxidant effects.
In the xanthinexanthine oxidase system estradiol (67.4 1.0%), 3a-sulfated-tibolone
(85.8 5.3%), 3a-17b-disulfated-tibolone (71.9 2.5%), 3b-sulfated-tibolone (73.9 5.0%),
and 3b-17b-disulfated-tibolone (65.8 3.4%) caused a significant decrease in superoxide
production. Conclusively, although tibolone itself did not show significant antioxidant capacity,
most of its active metabolites have antioxidant effects.

Antioxidant effect, estradiol, superoxide anion

inhibition, tibolone, tibolone metabolites

Introduction
Reactive oxygen species (ROS) are highly reactive oxygenderived metabolites that react with lipids, proteins, peptides and
nucleic acids. ROS from activated macrophages and neutrophils
enhance the intracellular signaling pathways of lymphocytes
and contribute to activation of the antigen-specific immune
response. Intracellular ROS are under the control of enzymes
like superoxide-dismutase, catalase and glutathione peroxidase.
If ROS reach extracellular space, the balance of free radicals
and antioxidant compounds is disturbed, because extracellular
scavengers are weaker than intracellular ones. There is a growing
awareness that oxidative stress plays a major role in various
clinical conditions like aging, diabetes, atherosclerosis, chronic
inflammation, malignant diseases, neurodegenerative diseases
and disorders associated with menopause [1,2].

*These authors contributed equally to this work.

Address for correspondence: Gabor Bekesi, MD, PhD, 2nd Department of
Internal Medicine, Faculty of Medicine, Semmelweis University,
Szentkiralyi u. 46, H-1088, Budapest, Hungary. Tel: +36/30/9324008.
Fax: +36/1/2660816. E-mail: bekesi.gabor@med.semmelweis-univ.hu
Szabolcs Varbiro, MD, PhD, 2nd Department of Obstetrics and
Gynecology, Faculty of Medicine, Semmelweis University, Ulloi ut 78/
A H-1082, Budapest, Hungary. Tel: +36/20/3359099. Fax: +36/1/
3346616. E-mail: varbiro.szabolcs@med.semmelweis-univ.hu

History
Received 9 April 2014
Revised 5 June 2014
Accepted 8 July 2014
Published online 23 July 2014

Estradiol has a known antioxidant effect through a direct

decrease in superoxide anion production, neutralization of excess
ROS and enhancement of cellular antioxidative defense molecules
(for review see [3]). Neutralization of excess ROS can be achieved
with certain structural features, such as the phenolic OH group
at the C-3 position of the A ring of the steroid molecule, and by
the activation of nitric oxide synthase (NOS) and nitric oxide
(NO) generation [3]. In our earlier study, as a result of the effect
of estrogen, increased myeloperoxidase activity and its negative
feedback, decreased superoxide anion levels were found [4].
Endogenous estrogen plays a major role in the protection of the
endothelial function against the effect of aging in premenopausal
women by preserving NO availability and eliminating oxidative
stress [5]. These effects may contribute to the well-known
advantages of premenopausal women compared to menopausal
women in respect of antioxidant status and prevention of the free
radical-mediated diseases mentioned above.
Upon the antioxidant effect of estrogen, we previously tested
the antioxidant capacity of many natural steroid hormones
and metabolites. In addition to estradiol, DHEA, DHEAS,
testosterone, cortisol, progesterone, estrone and estriol have
proven antioxidant activity. On the other hand, certain compounds (such as 11b-hydroxyprogesterone) had pro-oxidant
property [6,7].
After a systematic examination of the antioxidant effect of
natural steroid structures our aim is now to test synthetic steroid

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J. Stark et al.

structures commonly used in clinical practice. Tibolone is a

well known drug in menopausal medicine, relieves climacteric
symptoms and prevents postmenopausal bone loss. It is classified
as a selective tissue estrogenic activity regulator (STEAR) due
to its tissue-selective effects [8,9]. There is little information
so far about the effects of tibolone on superoxide production.
Some data are available about the influence of tibolone on
the NOSNO system: tibolone induced a sustained increase of
NO plasma levels in postmenopausal women, mainly through
activating NO synthesis in human endothelial cells [10,11].
In another study, tibolone was effective as an antioxidant in the
brain cortex, tibolone-treated animals showed lower lipid
hydroperoxide levels compared to the control ovariectomized
rats [12]. Tibolone and two of its metabolites, D4-tibolone and
3b-hydroxytibolone in a high concentration (10 6 M) increased
catalase activity in human breast cancer cells, thus the detoxification of hydrogen peroxide was enhanced [13].
The aim of our present study was to identify specific tibolone
metabolites, which may have an active effect on oxidative stress
and compare the effect of these compounds with known
antioxidant steroid structures.

Methods
Blood samples were obtained in EDTA tubes from 10 healthy
volunteers (women and men, aged 28 to 46). Volunteers were
medical doctors, all of whom gave informed consent. The study
was approved by the Semmelweis University ethical committee.
All volunteers had a negative medical history and none of
them was taking any medication. The blood was applied to
Ficoll in layers for the sedimentation of red blood cells and put
aside for an hour. Then it was transferred to 63 and 72% Percoll
and centrifuged for 25 min at a rate of 300  g at 20  C.
Granulocytes were separated as follows: they were bufferwashed twice and centrifuged with 220  g. Cells aggregated at
the bottom of the tube were re-suspended in a few milliliters of
buffer (Hanks salt solution, Biochrom KG, Berlin), then counted
using Turks solution. The cell concentration of the suspension
was adjusted to 5 million/ml. The granulocyte suspensions
were incubated for two hours at 37  C with different concentrations (10 7, 10 8 and 10 9 M) of steroid hormones. The
following steroids were tested in this study: estradiol (Sigma, St
Louis, MO), tibolone, 3a-hydroxytibolone, 3b-hydroxytibolone,
D4-tibolone, 3a-sulfated-tibolone, 3a-17b-disulfated-tibolone,
3b-sulfated-tibolone, 3b-17b-disulfated-tibolone (tibolone metabolites were provided by the manufacturer).

Gynecol Endocrinol, Early Online: 15

The superoxide anion generation of neutrophils was measured

using Guarnieris method [14], as modified by Jansson [15].
The quantity of free radicals released was assessed by
photometry; the reduction of ferricytochrome-C (Sigma, St
Louis, MO) by the superoxide was measured as optical density
at 550 nm. The maximum superoxide anion production was
assessed as change of optical density (DOD), five minutes after
stimulation with N-formyl-Met-Leu-Phe (FMLP, Sigma). The
final concentration of FMLP was 10 6 M. The result of the first
measurement just after adding FMLP to the cell suspension
served as the starting point (zero value). Further measurements
were carried out every minute (012345 min). Using the
molar extinction coefficient of cytochrome and standardizing the
output per 106 cells, results are given as nmol superoxide anion/
106 cells. Results for different steroids were given as percentages
of their controls in order to improve comparability. All compounds were tested with the same assay.
To control our results, we also tested the compounds in a noncellular superoxide generating system, the xanthinexanthine
oxidase reaction. During this additional experiment, we used 0.05
units/ml of xanthine oxidase (Sigma) in 100 mM K-phosphate
buffer, pH 7.8, containing 0.2 mM EDTA and 0.5 mM xanthine
(Sigma). Steroidal compounds were tested at 10 7 M.
Measurements of the reduction of ferricytochrome-C by
Guarnieris method (mentioned above) were carried out for
6 min at a wavelength of 550 nm [14].
Statistical analysis was performed using General Linear
Models (SAS 8.2 statistical software, Procedure GLM, Cary,
NC) and Dunnetts t test to compare each treatment versus
control. The comparisons for each compound were carried out on
the three concentration levels mentioned above. Results were
considered significant at the level of p50.05. Results are given as
means SEM. The error bars on the diagrams represent errors
between individuals.

Results
Ex vivo superoxide anion production after incubation
with different steroid concentrations
Granulocyte superoxide production was not different from the
control using 10 9 M of the various tibolone metabolites and the
reference estradiol.
At 10 8 M, estradiol (80.9 2.5%, p50.05), 3b-sulfatedtibolone (83.3 4.7%, p50.05), and 3b-17b-disulfated-tibolone
(81.0 4.2%, p50.05) caused a significant decrease in ex vivo
superoxide production (Figure 1).

Figure 1. Superoxide anion production of human neutrophil granulocytes in the percentages of controls in the presence of 10 8 M of different steroids
labeled in the figure. Results are given as means SEM. The error bars on the diagrams represent errors between individuals. White columns with an
asterisk show significant alterations compared to the controls (p50.05). Grey columns represent the non-significant groups.

DOI: 10.3109/09513590.2014.943727

At 10 7 M the above-mentioned compounds produced a

higher inhibition of superoxide formation and now the effect
of 3b-hydroxytibolone and 3a-sulfated-tibolone also became
significant: estradiol (76.4 4.2%, p50.001); 3b-hydroxytibolone
(82.9 5.3%, p50.05); 3a-sulfated-tibolone (81.1 4.4%,
p50.05);
3b-sulfated-tibolone
(79.2 5.7%,
p50.01);
3b-17b-disulfated-tibolone (74.6 5.1%, p50.0001) (Figure 2).
Tibolone, 3a-hydroxytibolone, D4-tibolone and 3a17b-disulfated-tibolone did not cause any significant
change in the superoxide production of neutrophil granulocytes at the concentrations used.

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Superoxide anion production in the xanthinexanthine

oxidase system
In the xanthinexanthine oxidase system estradiol (67.4 1.0%,
p50.05), 3a-sulfated-tibolone (85.8 5.3%, p50.05), 3a-17bdisulfated-tibolone (71.9 2.5%, p50.05), 3b-sulfated-tibolone
(73.9 5.0%,
p50.05)
and
3b-17b-disulfated-tibolone
(65.8 3.4%, p50.05) decreased superoxide production
significantly (Figure 3). Tibolone, 3a-hydroxytibolone, 3bhydroxytibolone and D4-tibolone did not cause any significant
change in superoxide production.

Discussion
In our earlier studies we found decreased superoxide anion
production after using several natural steroid hormones and their

Tibolone as antioxidant

intermediate metabolites [4,6,7,16]. Tibolone is rapidly metabolized into three steroid receptor activating compounds which
determine highly the biological activity in various tissues.
Tibolone metabolites are to a large degree sulfated and are
involved in the tissue selective action of tibolone [8,11,17].
High concentrations of sulfated metabolites are present in the
circulation (see references in [8]).
Sulfated metabolites are not active at the steroid receptor
level, but may have antioxidant properties, as we have shown.
Sulfconjugated catechol estrogens also have antioxidant activity
[18], and sulfated flavonoids possess this property as well [19].
DHEAS, the sulfated metabolite of DHEA also has antioxidant
properties [20]. The mechanism of action via a non-receptor path
is still unclear. Iwasaki et al. [20] suggested that the mechanism
may set in via the inhibition of proinflammatory cytokinestimulated, NF-kappa B-mediated transcription.
Some data are available on the improvement of free radicalmediated diseases after tibolone treatment. It has beneficial effect
on inflammation and oxidative state in postmenopausal women
[21]. Progression of Sjogrens syndrome might be prevented by
tibolone treatment [22]. The effect of tibolone on atherosclerosis
is somewhat contradictory: unlike estradiol, it decreases highdensity lipoprotein (HDL) cholesterol and triglycerides; however,
animal models and clinical studies did not confirm an increased
risk of plaque formation and cardiovascular diseases [8]. In a
study by Zandberg et al. [23] tibolone protected ovariectomized
and cholesterol-fed rabbits from atherosclerosis: it reduced the

Figure 2. Superoxide anion production of human neutrophil granulocytes in the percentages of controls in the presence of 10 7 M of different steroids
labeled in the figure. Results are given as means SEM. The error bars on the diagrams represent errors between individuals. White columns with an
asterisk show significant alterations compared to the controls (p50.05). Grey columns represent the non-significant groups.

Figure 3. Superoxide anion production in the xanthinexanthine oxidase system in the percentage of the controls in the presence of 10 7 M of different
steroids labeled in the figure. Results are given as means SEM. The error bars on the diagrams represent errors between individuals. White columns
with an asterisk show significant alterations compared to the controls (p50.05). Grey columns represent the non-significant groups.

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J. Stark et al.

accumulation of cholesterol, fatty streak formation, impairment of

endothelium-dependent response, and advanced lesion formation
after endothelial damage. The tibolone-derived prevention of
atherosclerotic lesion formation is in part carried out through
reducing leukocyte adhesion molecule expression on human
endothelial cells [24]. Beyond the effects on the NOSNO system
[1012], the antioxidant activity verified by this study may
explain the blocking effect of tibolone on these partially free
radical-mediated pathomechanisms.
Based on the results of the present study, we can conclude
that some of the tibolone metabolites have pronounced antioxidant activity. Beyond the steroid structure, we observed other
chemical specificities in this action. Almost all of the sulfated
metabolites display antioxidant activity, the strongest one was 3b17b-disulfated-tibolone in both tests used in this study. This
metabolite shows an activity which is comparable with the
reference compound estradiol and is present in large amounts in
the circulation of women who use tibolone [17]. Apparently,
phenolic A-ring and steroid receptor activity are not absolute
requirements for the antioxidant action of a compound. There is
no indication of any pro-oxidant activity. Both model systems ex
vivo and in vitro demonstrated similar effects of tibolone
derivatives. The concentrations used in this study are similar
to plasma levels of tibolone metabolites after tibolone treatment
of postmenopausal women [8,17], and were commonly used in
in vitro studies in the literature and in our own previous
measurements.
As mentioned earlier, oxidative stress plays a major role in
aging and subsequent disorders [1]. Diseases such as diabetes,
metabolic syndrome, atherosclerosis, hypertension and neurodegenerative diseases have well-established therapy. Some of these
medications have been found to have an antioxidant effect, such as
selegiline in Parkinsons disease [25], the angiotensin-converting
enzyme inhibitors captopril and ramipril [26,27], the nonselective beta-blocker carvedilol [28], and also some of the
statins and fibrates [29,30]. Tibolone has been used for the
treatment of menopausal complaints, and according to our
results, apart from these beneficial effects it also has an
antioxidant capacity through the active metabolites. Thus, it
might have some additive action combined with other first-line
drugs used for menopausal patients in different free radicalmediated illnesses. Our data may benefit the development of new
free radical scavengers/blockers using molecular design on the
target molecule.

Acknowledgements
The authors wish to express their gratitude to Organon
International Ltd. for their technical, material and methodological
support and for providing us with the different metabolites of tibolone,
to Balazs Gerecz for his administrative and technical support and to
Krisztina Nagy for her devoted efforts in solving technical problems in
laboratory work.

Declaration of interest
This study was sponsored by Organon International Ltd. and
Maecenator Foundation.

References
1. Gil del Valle L. Oxidative stress in aging: theoretical outcomes and
clinical evidences in humans. Biomed Aging Pathol 2011;1:17.
2. Babior BM. Phagocytes and oxidative stress. Am J Med 2000;109:
3344.
3. Kumar S, Lata K, Mukhopadhyay S, Mukherjee TK. Role of
estrogen receptors in pro-oxidative and anti-oxidative actions of
estrogens: a perspective. Biochim Biophys Acta 2010;1800:
112735.

Gynecol Endocrinol, Early Online: 15

4. Bekesi G, Kakucs R, Varbiro S, et al. In vitro effects of different

steroid hormones on superoxide anion production of human
neutrophil granulocytes. Steroids 2000;65:88994.
5. Viridis A, Taddei S. Endothelial aging and gender. Maturitas 2012;
71:32630.
6. Bekesi G, Racz K, Hrabak A, et al. Systematic investigation of
different steroid precursors with respect to their effect on superoxide
anion production by human neutrophil granulocytes. Horm Metab
Res 2004;36:15563.
7. Bekesi G, Tulassay Z, Racz K, et al. The effect of estrogens on
superoxide anion generation by human neutrophil granulocytes:
possible consequences of the antioxidant defense. Gynecol
Endocrinol 2007;23:4514.
8. Kloosterboer HJ. Historical milestones in the development of
tibolone (Livial ). Climacteric 2011;14:60921.
9. Biglia N, Maffei S, Lello S, Nappi RE. Tibolone in postmenopausal
women: a review based on recent randomised controlled clinical
trials. Gynecol Endocrinol 2010;26:80414.
10. Cicinelli E, Ignarro LJ, Galantino P, et al. Effects of tibolone on
plasma levels of nitric oxide in postmenopausal women. Fertil Steril
2002;78:4648.
11. Simoncini T, Mannella P, Fornari L, et al. Tibolone activates nitric
oxide synthesis in human endothelial cells. J Clin Endocrinol Metab
2004;89:4594600.
12. de Aguiar RB, Dickel OE, Cunha RW, et al. Estradiol valerate
and tibolone: effects upon brain oxidative stress and blood
biochemistry during aging in female rats. Biogerontology 2008;9:
28598.
13. Petit E, Courtin A, Kloosterboer HJ, et al. Progestins induce catalase
activities in breast cancer cells through PRB isoform: correlation
with cell growth inhibition. J Steroid Biochem Mol Biol 2009;115:
15360.
14. Guarnieri C, Melandri G, Caldarera I. Reduced oxidative activity of
circulating neotrophils in patients after myocardial infarction. Cell
Biochem Funct 1990;8:15762.
15. Jansson G. Oestrogen-induced enhancement of myeloperoxidase
activity in human polymorphonuclear leukocytes a possible cause
of oxidative stress in inflammatory cells. Free Radic Res Commun
1991;14:195208.
16. Bekesi G, Kakucs R, Sandor J, et al. Plasma concentration
of myeloperoxidase enzyme in pre- and postclimacterial
people: related superoxide anion generation. Exp Gerontol 2001;
37:13748.
17. Vos RM, Krebbers SF, Verhoeven CH, Delbressine LP. The in vivo
human metabolism of tibolone. Drug Metab Dispos 2002;30:
10612.
18. Takanashi K, Watanabe K, Yoshizawa I. On the inhibitory effect of
C17-sulfoconjugated catechol estrogens upon lipid peroxidation of
rat liver microsomes. Biol Pharm Bull 1995;18:11205.
19. Lodi F, Jimenez R, Moreno L, et al. Glucuronidated and sulfated
metabolites of the flavonoid quercetin prevent endothelial dysfunction but lack direct vasorelaxant effects in rat aorta. Atherosclerosis
2009;204:349.
20. Iwasaki Y, Asai M, Yoshida M, et al. Dehydroepiandrosteronesulfate inhibits nuclear factor-kappaB-dependent transcription in
hepatocytes, possibly through antioxidant effect. J Clin Endocrinol
Metab 2004;89:344954.
21. Vassalle C, Cicinelli E, Lello S, et al. Effects of menopause and
tibolone on different cardiovascular biomarkers in healthy women.
Gynecol Endocrinol 2011;27:1639.
22. Sartore A, Grimaldi E, Guaschino S. The treatment of Sjogrens
syndrome with tibolone: a case report. Am J Obstet Gynecol 2003;
189:894.
23. Zandberg P, Peters JLM, Demacker PNM, et al. Tibolone
prevents atherosclerotic lesion formation in cholesterol-fed, ovariectomized rabbits. Arterioscler Thromb Vasc Biol 1998;18:
184454.
24. Sator K, Sator MO, Sator PG, et al. Effects of tibolone on selectins
in postmenopausal women. Maturitas 2006;53:16670.
25. Foley P, Gerlach M, Youdim MB, Riederer P. MAO-B inhibitors:
multiple roles in the therapy of neurodegenerative disorders?
Parkinsonism Relat Disord 2000;6:2547.
26. Benzie IF, Tomlinson B. Antioxidant power of angiotensinconverting enzyme inhibitors in vitro. Br J Clin Pharmacol 1998;
45:1689.

DOI: 10.3109/09513590.2014.943727

Gynecol Endocrinol Downloaded from informahealthcare.com by Michigan University on 10/24/14

For personal use only.

27. Keles MS, Bayir Y, Suleyman H, Halici Z. Investigation of effects of

Lacidipine, Ramipril and Valsartan on DNA damage and oxidative
stress occurred in acute and chronic periods following isoproterenolinduced myocardial infarct in rats. Mol Cell Biochem 2009;328:
10917.
28. Gasparova Z, Ondrejickova O, Gajdoskova A, et al. Oxidative stress
induced by the Fe/ascorbic acid system or model ischemia in vitro:

Tibolone as antioxidant

effect of carvedilol and pyridoindole antioxidant SMe1EC2 in young

and adult rat brain tissue. Interdiscip Toxicol 2010;3:1226.
29. Murrow JR, Sher S, Ali S, et al. The differential effect of statins on
oxidative stress and endothelial function: atorvastatin versus
pravastatin. J Clin Lipidol 2012;6:429.
30. Heller FR, Descamps O, Hondekijn JC. LDL oxidation: therapeutic
perspectives. Atherosclerosis 1998;137:S2531.