In this cross-sectional study among postmenopausal Japanese women, we found no statistically significant association between endogenous sex hormone level and the global methylation level of peripheral blood leukocyte DNA. To our knowledge, this is the first study to test the hypothesis that endogenous sex hormones are potential determinants of the global methylation level of leukocyte DNA. Our findings did not support this hypothesis, however, and suggest that endogenous sex hormones might not be major determinants.
Many epidemiological studies have confirmed the important role of endogenous sex hormones, particularly estrogens, in the etiology of breast cancer [11
]. A pooled analysis of nine prospective studies showed that higher estrogens and their androgen precursors were associated with a higher risk of breast cancer in postmenopausal women [13
]. In addition, several well-known risk factors have also implicated an etiologic role of sex hormones, including age at menarche and menopause, parity and age at first birth, and BMI [11
]. Recently, Choi et al.. reported that global hypomethylation was associated with an increased risk of breast cancer [8
], suggesting that the global methylation level of leukocyte DNA plays a role in the etiology of breast cancer. However, our findings suggest that global methylation level does not serve as a surrogate marker for the level of endogenous sex hormones and that the mechanisms by which global hypomethylation increases the risk of breast cancer might involve non-hormonal factors.
Several possible explanations for the observed absence of associations can be considered. First, misclassification due to inaccurate measurement may have attenuated the true association. However, since the reproducibility of assays for global methylation and sex hormone levels were relatively high, with an intra-assay CV for global methylation of 6.4%, and a range for hormones from 4.5% for testosterone to 10.6% for bioavailable estradiol, the absence of associations was unlikely due to measurement error. On the other hand, global methylation level might be affected by differences in the proportion of white blood cell types. Indeed, Zhu et al.. reported that the proportion of neutrophils and lymphocytes among white blood cell types modified LINE-1 methylation levels measured in blood DNA [9
]. In the present study, since we measured global methylation level on white blood cells by the LUMA method, the possibility of measurement error due to different proportions of white blood cell types cannot be excluded. Such misclassification might accordingly have attenuated the true association, which might in turn have explained our results, at least in part. Second, the single time point measurement of serum hormone levels might not have captured the overall activity of the sex hormone-related signaling pathway of the subject up to the age of study. Third, although the present study included a total of 185 women, it may not have had sufficient statistical power to detect small associations.
Differences in assessment methods of global DNA methylation require consideration. While several previous studies estimated global DNA methylation based on data restricted to repetitive DNA elements, such as Alu and LINE-1 [7
], the present study used LUMA, which allows the estimation of genome-wide DNA methylation levels. Differences in assessment methods might therefore have affected study results. For example, Choi et al.. found that global hypomethylation of leukocyte DNA measured by 5-methyldeoxycytosine (5-mdC) was associated with an increased risk of breast cancer but saw no association with the LINE-1 methylation level of leukocyte DNA [8
]. Similarly, Wischwendter et al. reported no association between the Alu methylation level of leukocyte DNA and breast cancer risk [7
The mechanisms by which endogenous sex hormone levels might affect global methylation status have not been elucidated. However, there is some evidence that estrogens or endocrine disruptors might alter DNA methylation status [24
]. Studies in mice have shown that neonatal exposure to diethylstilbestrol (DES), a synthetic non-steroidal estrogen, induced hypomethylation of estrogen-responsive lactoferrin gene and proto-oncogene c-fos
in utero [24
]. Another study showed that exposure to persistent organic pollutants such as polychlorinated biphenyls, many of which are known endocrine disruptors, was associated with global DNA hypomethylation in Greenlandic Inuit [26
In our subjects, subgroup analyses suggested that alcohol drinking modified the association between endogenous sex hormone levels and the global methylation level of leukocyte DNA: higher DHEAS was significantly associated with a lower level of global methylation among alcohol drinkers, whereas no association was observed among non-drinkers. Since alcohol consumption interferes with folate metabolism [27
] and decreases levels of serum folate [28
], our findings might indicate that higher sex hormone levels were associated with a lower level of global methylation among subgroups with a low folate status only. This hypothesis, however, does not appear to be supported by our subgroup analyses by folate intake or MTHFR
polymorphisms (rs1801133 and rs1801131). Although the T allele of MTHFR
(rs1801133) and C allele of MTHFR
(rs1801131) have shown lower enzyme activity [29
], no remarkable difference in global methylation level was observed among genotypes of MTHFR
. These inconsistent findings might be explained by multiple comparisons, insufficient power, or the possibility that alcohol consumption interacts with sex hormone levels via a mechanism other than the folate-related metabolism. In any case, the findings of our subgroup analyses should be interpreted with caution, and further research to clarify effect modification by dietary and genetic factors related to one-carbon metabolism on the association between sex hormone level and global methylation level is clearly required.