Numerous reports have attested to the health damaging effects of red wine and its components beyond excess alcohol consumption, for example - owing to pesticide and heavy metal content 1 2 . In contrast, many reports point to the health protective effects of red wine owing to the abundance of anti-oxidants 3 4 . Beyond modulating oxidative damage, one focus has been on the female endocrine system, following the reports that red wine has anti-aromatase properties 5 . This discovery broadened the debate regarding the link between alcohol intake and risk of developing breast cancer 6 7 .

Equally, the associations of high and low testosterone levels with the development of various forms of prostate cancer have been subjected to considerable debate 8 9 10 . Given the inhibitory effects of red wine on aromatase it is conceivable that red wine also affects aspects of testosterone metabolism. Although recent epidemiological studies have suggested red wine consumption is not a potential risk factor for prostate cancer 11 12 , the effects of red wine on testosterone metabolism warrant investigation.

Glucuronidation is a major metabolic pathway for the elimination of testosterone and numerous compounds from the body 13 14 . Glucuronidation of testosterone involves the transfer of a glucuronosyl group from UDP-glucuronic acid (UDPGA) to form the steroid conjugate, testosterone glucuronide, which is then excreted in urine 13 . The UDP-glucuronosyltranferase UGT2B17 enzyme is the main steroid glucuronidation enzyme of the UGT isotopes with more than double the glucuronidation activity compared to the second most active enzyme involved in glucuronidation of testosterone, UGT2A1 15 .

The metabolism of testosterone by UGT2B17 has been shown to differ between individuals owing to the variations in the expression of UGT2B17, which has been found to alter with ethnicity, affecting the excreted steroid concentrations 16 . In in vitro studies, the rate of testosterone glucuronidation has also been shown to be reduced with inhibitors of UGT2B17, such as non-steroidal anti-inflammatory drugs 15 . Whilst various drugs and compounds are glucuronidated as a substrate and inhibit UGT2B17 13 , little is known about the inhibitory effects common dietary substances could have on UGT2B17 and testosterone glucuronidation.

Recently, green and white teas and purified catechin constituents have been shown to inhibit the key testosterone glucuronidation enzyme UGT2B17 in a supersome-based assay 17 . Red wine is another rich source of phenolic compounds that have been found to exert anti-oxidant health benefits in humans 18 . Given the inhibitory effects of green and white tea on UGT2B17, along with the debate on red wine and prostate cancer, it is timely to investigate if phenolic compounds in red wine have an inhibitory effect on testosterone metabolism and excretion.

The aim of this study was to analyze the inhibitory effects of a dietary red wine sample and the common phenolic compounds found in red wine, independent of the effects of alcohol, on the glucuronidation of testosterone through the inhibition of UGT2B17. A further aim was to study the potential inhibitory effect of the common wine by-product 4-ethylphenol on testosterone glucuronidation.

This report extends the previous study which demonstrated that tea and its component flavones competitively inhibit testosterone glucuronidation by UGT2B17. The results of this study showed that phenolic compounds commonly found in red wine, but also in many other foods, have comparable effect on testosterone glucuronidation. The rate of glucuronidation was similar on addition of the wine sample once the ethanol had been removed, indicating that it was likely to be the phenolic compounds that caused inhibition. Further studies revealed that ethanol had no effect at the concentrations found in the added wine sample. However, at higher concentrations of ethanol (>1%) the UGT2B17 enzyme activity was reduced. Ethanol has been linked to increased testosterone and aggression in male hamsters 21 and increased testosterone in rat brain 22 . From our results, the effects of alcohol on UGT2B17 are unlikely to account for the increase in testosterone, unless extremely high doses are consumed.

Several of the individual wine phenolic compounds inhibited the glucuronidation of testosterone at different efficiencies. The maximum inhibition was observed for quercetin, followed by 4-ethylphenol and caffeic acid. The serum concentrations of phenolic compounds that are commonly found in wine can be increased through supplementation such as with quercetin 23 . In one study, after supplementation, 1.5 μM quercetin levels in plasma were reported 24 . This concentration of quercetin affected a 9% reduction in UGT2B17 activity despite a high concentration of testosterone at 20 μM. The reported mean level of serum testosterone in adult males is 35.9 nM 25 . Given the inhibition is competitive; these much lower concentrations of testosterone should result in higher inhibition of UGT2B17 by quercetin. Future studies are warranted to investigate the effects of red wine and its components at physiological levels of testosterone.

In addition, plasma concentrations of caffeic acid have been shown to increase after the consumption of red wine 26 . Given these increases in serum concentrations through supplementation it may be possible to further reduce testosterone metabolism through glucuronidation.

The phenolic compound 4-ethylphenol has been found in red wines to produce distinct aromas 27 , as well as being produced by the spoilage of yeast, hence it can be found in other dietary substances 28 and enters knowingly or unknowingly into the diet. 4-Ethylphenol has also been shown here to inhibit the glucuronidation of testosterone through the UGT2B17 enzyme. The inhibition of UGT2B17 by 4-Ethylphenol was found to be greater at the lower 50 μM level of testosterone. However, at high initial testosterone concentrations above 150 μM there was very little or no inhibition showing that increasing the concentration of testosterone will overcome the inhibiting compound. 4-Ethylphenol has been shown to be a substrate for UGT2B17 with a glucuronide being formed 13 ; therefore it is likely that these compounds are acting competitively with the UGT2B17 enzyme.

Whilst it has been found that no correlation exists between the variations of the UGT2B17 genotype with alterations in circulating serum testosterone levels 29 30 31 , another study has found significant differences in circulating testosterone with an increase in individuals expressing no copy number of UGT2B17 compared to individuals with one or two copies of UGT2B17 32 . It has yet to be determined if any direct inhibition of steroid glucuronidation enzymes could alter the levels of circulating serum testosterone in addition to altering the levels of testosterone excreted in urine. These results augment the previous study revealing that tea catechins can inhibit testosterone metabolism by supersomes containing UGT2B17 17 . The ubiquitous presence of quercetin and other active flavonols, along with the catechins, in many foodstuffs indicate that any in vivo effects may be common. These effects, if found to occur in vivo, may have a pronounced effect on people with endocrine disorders or very low levels of endogenous testosterone, owing to high levels of receptor expression to compensate. A further aspect, although not studied here, is the potential interaction of quercetin-containing foodstuffs with drug metabolism as some drugs are metabolized via the action of UGT2B17 33 .

In conclusion it has been found that a commonly consumed dietary substance, red wine along with phenolic compounds present in red wine, inhibit testosterone glucuronidation. These results have also shown that although some of these compounds are not substrates of UGT2B17 they can inhibit the enzyme in supersomes.

The authors declare that they have no competing interests.

All authors contributed to the study design, data analyses and manuscript preparation. CJ completed the laboratory studies. All authors approved the final manuscript.