Elsevier

Steroids

Volume 76, Issue 12, November 2011, Pages 1400-1406
Steroids

Short communication
Relevance of the selective oestrogen receptor modulators tamoxifen, toremifene and clomiphene in doping field: Endogenous steroids urinary profile after multiple oral doses

https://doi.org/10.1016/j.steroids.2011.06.005Get rights and content

Abstract

The present study was performed to investigate the influence of the intake of selective oestrogen receptor modulators on the urinary endogenous steroids profile. For this purpose the circadian variability of luteinizing hormone, follicle-stimulating hormone, testosterone, 5α-androstan-3α,17β-diol, 5β-androstan-3α,17β-diol, epitestosterone, 4-androstenedione, androsterone and etiocholanolone were measured on eight subjects (four males and four females) by gas chromatography–mass spectrometry and chemiluminescent immunometric assay techniques before and after oral administration of multiple doses of either tamoxifen (80 mg for 2 days) or toremifene (120 mg for 2 days) or clomiphene (100 mg for 2 days). The individual baseline variability of the steroids studied was set up by collecting the urine samples every 3 h, for 3 days prior to the treatment; whereas the evaluation of the effects of the oral administration of multiple doses of selective oestrogen receptor modulators on the steroid urinary profile was assessed by collecting urine samples every three hours for at least five days from the first administration.

The results of our measurements showed that, only in male subjects, the relative urinary concentrations of testosterone, epitestosterone and 4-androstenedione were significantly altered generally after the second day of drug administration. While no significant effects were recorded in both sexes on the luteinizing hormone, follicle-stimulating hormone, androsterone, etiocholanolone, 5α-androstan-3α,17β-diol and 5β-androstan-3α,17β-diol urinary levels and on testosterone/epitestosterone, 5α-androstan-3α,17β-diol/5β-androstan-3α,17β-diol and androsterone/etiocholanolone ratios.

Highlights

▸ Since 2005, SERMs were in the World Anti-Doping Agency list of prohibited substances and methods. ▸ The effects of SERMs administration on the sport performance and on the normal circadian fluctuation of the endogenous hormonal parameters are still unknown. ▸ The purpose of this study was to verify whatever the oral administration of SERMs (clomiphene, toremifene and tamoxifen) leads to significant variations on the endogenous androgenic steroid urinary excretion. ▸ The results obtained on urine samples show that only in male subjects, the relative urinary concentrations values of testosterone were significantly altered generally after the second day of drug administration. ▸ The same finding was not observed in women, whose testosterone is derived mainly from extra-gonad sources that are not involved in any homoeostatic feedback regulation.

Introduction

According to the annual statistic data of the World Anti-Doping Agency (WADA) based on the accredited laboratories reporting [1], anabolic androgenic steroids are still the most frequently abused drugs used in sports. Nevertheless, the evolution of the analytical technologies has led the athletes to use a variety of indirect strategies to circumvent the ban of direct androgen doping, such as, for example, the indirect stimulation of hypothalamic gonadotropin-releasing-hormone (GnRH) secretion by increasing endogenous luteinizing hormone [2], [3], [4]. One of the factors known to enhance endogenous hypothalamic GnRH secretion includes the use of selective oestrogen receptor modulators (SERMs) that binding competitively to oestrogens hypothalamic and pituitary receptors block sex steroid negative feedback. Previous researchers demonstrated that the administration of these agents increases the blood testosterone levels in men [3], [4]. The same finding was not observed in women, because in contrast to men in whom blood testosterone concentrations are strictly controlled by negative feedback regulation of the hypothalamic–pituitary–gonad axis, in women blood testosterone is derived mainly from extra-gonad sources that are not involved in any homoeostatic feedback regulation [2], [3], [4].

Since 2005, SERMs are included, with other similar drugs, in the Section 4 “agents with anti-oestrogenic activity” of the WADA list of prohibited substances and methods [5]. The athletes could illicitly use SERMs for two main reasons: (i) to increase endogenous testosterone levels, with the aim to by-pass the specific testing regimens for known synthetic androgens including exogenous testosterone, and (ii) to balance the adverse effects of an extensive abuse of anabolic androgenic steroids [3], [4]. Little is known about the effect of SERMs administration on the sport performance and on their possible interaction with the normal circadian fluctuation of the endogenous hormonal parameters that are presently selected as markers of an atypical urinary steroid profile in the doping field [6]. The purpose of this study was to verify whether the oral administration of multiple doses of SERMs (tamoxifen, toremifene or clomiphene) leads to significant variations on testosterone, epitestosterone, 4-androstenedione, androsterone, etiocholanolone, 5α-androstan-3α,17β-diol, 5β-androstan-3α,17β-diol, luteinizing hormone and follicle-stimulating hormone urinary concentration. For this purpose the steroids urinary levels measured before the oral administration of multiple doses of tamoxifen, toremifene or clomiphene were compared with those obtained after administration of the drug(s) using the Student t-test (p < 0.05). The data obtained were also evaluated taking into account the procedure currently adopted by the WADA to give an atypical or a positive result in case of longitudinal studies evaluation (CV% ⩾30 for males and CV% ⩾60 for females). Specifically for each subject the coefficient of variation was calculated considering all the measurements of the samples obtained before and after SERMs administration. In addition the mean concentrations value obtained before drug administration (individual reference baseline value) were compared with every single value obtained after the administration [6].

Section snippets

Standards, chemicals and reagents

Tamoxifen (Nolvadex®) was purchased from AstraZeneca (Milan, Italy). Toremifene (Fareston®) was purchased from Orion Pharma (Milan, Italy). Clomiphene (Clomid®) was purchased from Bruno Farmaceutici S.p.A. (Rome, Italy).

Androsterone and etiocholanolone were supplied by Sigma–Aldrich (Milan, Italy).

Testosterone, 5α-androstan-3α,17β-diol, 5β-androstan-3α,17β-diol, epitestosterone, 4-androstenedione, deuterated testosterone, deuterated epitestosterone, deuterated androsterone and deuterated

Individual baseline

Fig. 1A, Fig. 1B, Fig. 1C, Fig. 2A, Fig. 2B, Fig. 2C show that in both sexes the excretion of testosterone and epitestosterone was maximal in the morning and decreased along the day reaching a minimum value in the evening. The same circadian trend was registered for 4-androstenedione, 5α-androstan-3α,17β-diol, 5β-androstan-3α,17β-diol, androsterone and etiocholanolone (data not shown). The variation in T/E ratio along the day was less than 20% in both males and females (see Fig. 1A, Fig. 1B,

Discussion and conclusions

At present, and to the best of our knowledge, little is known about the effect of the administration of SERMs on the normal circadian fluctuation of the endogenous hormonal parameters that are presently selected as markers of an atypical urinary steroid profile in doping field [6]. Data collected in this study have shown that in male subjects the relative urinary concentrations values of testosterone were significantly altered generally the second day of oral administration of multiple doses of

Acknowledgement

This work has been supported in part by a Research Grant of the Italian Department of Health (“Ministero della Salute, Commissione per la vigilanza sul doping e sulla tutela sanitaria delle attività sportive”).

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