In this pilot study we evaluated the association of 15 urinary estrogen metabolites, quantified by LC-MS, with prostate cancer risk. We observed a suggestive trend toward decreased urinary concentration of metabolites with high estrogenic activity, namely16-KE2 and 17-epiE3, among prostate cancer patients. Our analysis confirms that smoking is a modifier of urinary estrogen levels. Although needing confirmation in larger studies, our study shows that it is possible to detect a multitude of estrogen metabolites as well as differences in estrogen metabolites in urine between prostate cancer cases and controls.
Urine contains both biologically active estrogens, which includes unconjugated parent estrogens, their phase I metabolites, and O-methylated catechol estrogens, as well as biologically inactive estrogens, which includes sulfate and/or glucuronide conjugates. Because estrogen metabolites are mostly present in urine as glucuronide or sulfate conjugates, we measured total estrogen metabolites in men which is the sum of the glucuronidated, sulfated and unconjugated forms of each estrogen metabolite [29
Both 16-KE2 and 17-epiE3 are products of the 16α-hydroxylation pathway and derive from the biologically active 16α-OHE1 metabolite (). One of the leading hypotheses that explains the role of estrogens in breast carcinogenesis contradicts our observations of a potential protective effect of 16-KE2 and 17-epiE3 in prostate cancer. According to the breast cancer hypothesis, 16α-hydroxylated estrogens induces breast carcinogenesis due to their much stronger hormonal and mitogenic activity as compared to the catechol estrogens [30
]. However, it is possible that estrogen metabolites differentially affect different organs and thus extending results from breast to prostate may be misleading. For instance, both 16-KE2 and 17-epiE3 have shown a preferential binding capacity for ERβ [7
] which was shown to have a protective role within the prostate [9
]. We should also note that although 17-epiE3 retains close to 75% of the estrogenic activity of the E2, 16-KE2 has a significantly reduced binding affinity to ERα receptor and to a lesser extend to the ERβ receptor [7
]. Furthermore, we cannot rule out the possibility that the observed protective effects of these estrogen metabolites could be by chance. This may be particularly true for the low abundance 17-epiE3 for which the case control differences did not persist in our adjusted multivariate analysis. In this study we did not examine the androgen to estrogen balance and its potential association with prostate cancer risk. Androgens and estrogens are transported in the circulation by the sex hormone-binding globulin (SHBG). As shown previously [31
], controlling for serum SHBG levels may be necessary in order to find the relevant association between sex hormone levels and prostate cancer risk. Thus, larger studies that measure androgens as well as SHBG are needed to confirm our findings.
The 2-OHE1/16α-OHE1 ratio has been used as an index of the relative strengths of the two competing oxidative pathways in breast cancer with higher ratios having been associated with a reduced risk for the disease [32
]. Our observation that smoking alters the concentrations of the less estrogenic 2-OHE1 metabolite and marginally increases the 2-OHE1/16α-OHE1 ratio in urine suggests the anti-estrogenic effects of smoking [33
] and raises the issue of the role of smoking in prostate cancer development. Our finding contradicts that of Muti et al.
who did not observe an association between smoking status and urinary 2-OHE1 but reported increased levels of urinary16α-OHE1 among current smokers compared to never/former smokers [20
]. The different methodologies used in our study compared to that by Muti and colleagues could explain partly the contradictory findings. Additionally, while only patients with clinically apparent disease (stage B and higher) were included in the study by Muti and colleagues, most of the prostate cancer cases in the current study were of early disease stages.
In this study, the ranking of estrogens and its metabolites with respect to urinary concentrations was similar among the study groups. The estrogen metabolite 4-OHE1 ranked slightly higher among cases than the 2 control groups; however, there was no difference in the median concentration of the metabolite among the 3 groups. Given the rapid metabolic clearance of catechol estrogens, it is hard to estimate the contribution of 4-OHE1 to cancer risk. Animal studies have shown that exposure to 4-OHE1 and 4-OHE2 leads to formation of catechol estrogen quinones (CEQ) and subsequently depurinating DNA adducts, a process that is a putative tumor initiating event [34
]. In humans, CEQ-derived DNA adducts are present in urine samples from subjects with prostate cancer in higher amounts compared to controls [23
]. Based on this principle, low concentrations of 4-OHE1 would result in less adduct formation and would be protective against prostate cancer. However, it has also been postulated that 2-pathway catechol estrogens may actually be protective since their formation precludes 16-hydroxylation [37
Of interest is our observation that there was higher (abnormal) protein-to-creatinine ratio in the biopsy control group compared to both the healthy and prostate cancer groups. Increased protein-to-creatinine ratio is associated with proteinuria which is subsequently associated with renal function. Urinary excretion of endogenous and exogenous compounds is determined by glomerular filtration and tubular secretion. Although we could not confirm renal abnormalities as a diagnosis for the subjects that are in the biopsy control group, our observations prompted us to investigate abnormal protein-to-creatinine ratio in relation to estrogen concentrations. Analysis among the healthy control group with signs of proteinuria revealed an association with elevated 2-and 4- methoxyestrogens (2-MeOE1, 4-MeOE1, 2-MeOE2, 4-MeOE2). Methoxyestrogens are considered as potential therapeutic agents due to their antitumor activity via induction of apoptosis and inhibition of angiogenesis [38
]. The anti-proliferative effect of methoxyestrogens has not yet been demonstrated in relation to the prostate but it was shown that in breast cancer cells the effect can occur independently of ERα and ERβ [41
]. Similar observations were made in other cells such as pancreas, leukemia and lung [42
It is difficult to establish a temporal relation between urinary concentration of estrogens and prostate cancer risk in a cross-sectional study. The mean preclinical duration for prostate cancer has been estimated at least as a decade [44
]. A limitation of our study is that urine samples were collected throughout the day; a 24-hour urine collection would be ideal. Although circadian variations of plasma testosterone and estrogens have been demonstrated among younger men [45
], a recent study showed that testosterone concentrations in older men (mean age 60 years) are stable throughout the morning and early afternoon, declining modestly thereafter [46
]. The results imply that the diurnal variation of androgens in our age group (mean age 63 years) is a minor concern. We are not aware of a similar study of estrogens but similarity of the pathways would suggest that they might follow the same trends. We adjusted for time of collection in our final model but residual confounding could bias the study towards the null.
In summary, we evaluated the association between 15 estrogen metabolites and prostate cancer risk in a small case-control study. The results show only modest differences. We observed a tendency for lower urinary concentration of the 16-KE2 and 17-epiE3, metabolites with high estrogenic activity, among prostate cancer patients. Larger studies are needed to confirm these findings that also account for androgen levels and SHBG. In addition, a longitudinal study would likely be a better design to improve the assessment of the potential long term effects of estrogen metabolites on prostatic carcinogenesis.