Non-steroidal anti-inflammatory drugs interact with testosterone glucuronidation
Introduction
Testosterone and epitestosterone are excreted to the urine mainly as glucuronide conjugates, the formation of which is catalyzed by UDP-glucuronosyltransferases (UGTs). The UGTs are membrane bound enzymes of the endoplasmic reticulum that play important roles in the metabolism of drugs and other foreign compounds, and in the metabolism of endogenous compounds such as bilirubin and steroid hormones [1], [2]. The human UGTs are divided into several subfamilies, the most studied of which are 1A and 2B [3]. The UGTs 2B7, 2B15, and 2B17 are the principal catalysts of androgen glucuronidation in humans [4], [5]. UGT2B17 was originally identified as the main enzyme in testosterone glucuronidation in vivo since homozygous deletion of the gene encoding UGT2B17 (del/del) is associated with low or negligible urinary excretion of testosterone glucuronide [6]. An in vitro study showed that UGT2B15 and UGT2B17 are the major enzymes in human liver microsomal (HLM) testosterone glucuronidation [7]. Recent screening of all the human UGTs of subfamilies 1A, 2A and 2B for testosterone glucuronidation activity further demonstrated that UGT2B17 is by far the most important enzyme in testosterone glucuronidation, whereas UGT2B7 plays a correspondingly important role for epitestosterone glucuronidation [8], [5]. In subjects devoid of a functional UGT2B17 (del/del), UGT2B15 may be the main contributor to the residual testosterone glucuronidation activity. It was recently shown that UGT2B17 del/del individuals have higher relative mRNA levels of UGT2B15 in the liver compared to those carrying the UGT2B17 ins/ins genotype [9]. Further, the functional UGT2B15 Tyr85Asp polymorphism was associated with urinary levels of testosterone metabolites in UGT2B17 del/del individuals.
Testosterone is sometimes abused by athletes and the current method to detect possible illicit intake of testosterone is based on determination of the urinary ratio of testosterone glucuronide and epitestosterone glucuronide, often referred to as the T/E [10]. When the T/E is above 4.0, doping in sports is suspected [11] and such samples must be subjected to further analyses for determining if the testosterone was of endogenous or exogenous origin. The cut-off value of 4.0 for the T/E serves reasonably well for people that have a functional UGT2B17. However, administration of exogenous testosterone to individuals devoid of the UGT2B17 gene, rarely yields T/E values that exceed this cut-off ratio [12]. Because of this, future test programs may use Bayesian individual-based, rather than population-based, T/E cut-off ratios [13]. It has been shown that inclusion of the UGT2B17 genotype information in the Bayesian model, markedly improved the sensitivity of the test [14]. Nonetheless, the current approved method still relies on the cut-off value of 4.0 and it is important to clarify factors that affect it.
The UGTs are involved in the metabolism of a variety of compounds, including several non-steroidal anti-inflammatory drugs (NSAIDs) [15]. These drugs are used in the treatment of pain and inflammation, and they are commonly taken by athletes [16]. Many NSAIDs are metabolized through glucuronidation and UGT2B7 has previously been identified as the major enzyme in NSAID glucuronidation [17], [18], [1], [15]. UGT2B7, however, is not the only human UGT that catalyzes NSAIDs glucuronidation and other UGTs possess high activity towards some drugs of this group, including UGT1A1 for sulindac sulfonate, UGT1A3 for flurbiprofen, sulindac sulfonate and diclofenac, UGT1A9 for indomethacin, and UGT2B17 for diclofenac [15].
Since both steroids and NSAIDs are substrates of UGTs, we have postulated that testosterone and/or epitestosterone glucuronide formation may be affected by the co-use of NSAIDs. To examine this hypothesis, we have studied the interaction of diclofenac and ibuprofen with testosterone glucuronidation in human liver microsomes and recombinant UGTs.
Section snippets
Materials
Testosterone (4-androsten-17β-ol-3-one), diclofenac sodium salt, Brij®58P, and uridine-5-diphospho glucuronic acid triammonium salt (UDPGA) were purchased from Sigma (Sigma–Aldrich Chemie GmbH, Steinheim, Germany). Ibuprofen was from Orion Pharma (Espoo, Finland). Testosterone glucuronide was from Steraloids Inc. (Newport, RI, USA). Magnesium chloride hexahydrate, disodium hydrogen phosphate, and potassium dihydrogen phosphate were from Merck (Darmstadt, Germany). Dimethyl sulfoxide (DMSO) and
Inhibition studies in HLM
The effects of diclofenac and ibuprofen on the glucuronidation of testosterone and epitestosterone in human liver microsomes are shown in Fig. 1. Both NSAIDs inhibited testosterone glucuronidation, albeit at different efficiencies. The mean IC50 value for diclofenac was 64 μM, whereas for ibuprofen it was 213 μM (Table 1). A minor inhibition of epitestosterone glucuronidation by diclofenac was also observed, but the IC50 value was very high, 1.06 mM, rendering it non-significant with respect to
Discussion
Here we show for the first time that both diclofenac and ibuprofen inhibit testosterone glucuronidation in human liver microsomes. On the other hand, the inhibition of epitestosterone glucuronidation in HLM by either drug was minor and only at very high concentration (Fig. 1). The latter results were somewhat unexpected since epitestosterone is mainly glucuronidated by UGT2B7 [8], [5] an enzyme that readily glucuronidates both diclofenac and ibuprofen [15]. Moreover, the morphine and zidovudine
Acknowledgements
We gratefully acknowledge the scientific expertise and technical assistance of Mika Kurkela. The skilful technical assistance of Inkeri Huttunen, Johanna Mosorin and Sanna Sistonen is acknowledged. Waters Co., Finland is thanked for providing us with the UPLC Acquity device, and United Laboratories Ltd., Helsinki, Finland, for providing us with the analytical glucuronide standards. This work was supported by the World Anti Doping Agency (WADA), Karolinska Institutet funds and the Swedish
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