Estrogen replacement therapy increases genomic DNA methylation status in postmenopausal women
aDepartment of Clinical and Experimental Medicine, University of Verona School of Medicine, Verona, Italy, bLipid Metabolism Laboratory, cVitamins and Carcinogenesis Laboratory; Jean Mayer USDA Human Nutrition Research Center on Aging at Tufts University, Boston, Massachusetts, United States
AIM: Both epidemiologic and cell biology studies have demonstrated that hormonal replacement therapy (HRT) has important relationships with the risk for major cancers. HRT appears to contribute to the development of breast, endometrial and ovarian cancers, but significantly reduces the incidence of colorectal cancer. However, decades of mechanistic studies can neither fully explain these observations nor define the chemopreventive values of estrogen therapy. Since previous studies have suggested that higher estrogen levels are inversely related to serum total homocysteine (tHcy) concentrations, which are known to affect tissue DNA methylation status, we investigated whether decreased tHcy status due to estrogen therapy can alter genomic DNA methylation status and whether genomic DNA methylation status may represent one of estrogen therapeutic effects. METHODS: Thirteen healthy postmenopausal women (age 57 ± 6 years and weight 69.9 ± 12.3 kg) were enrolled in a double-blind, placebo-controlled, randomized, crossover study comparing, in two different phases, the effects of conjugate equine estrogen (CEE, 0.625 mg/day) versus placebo. Each phase lasted 8 weeks and the phases were separated by a 4-week washout period. At 8 week of each phase, blood was drawn to measure plasma tHcy and pyridoxal-5'-phosphate (PLP, vitamin B6) and serum folate/vitamin B12. DNA was extracted from peripheral mononuclear cells to measure the genomic DNA methylation status. Plasma tHcy was determined by HPLC with fluorimetric detection. Plasma PLP was determined by the tyrosine decarboxylase apoenzyme method. Serum folate and vitamin B12 concentrations were determined by a radioimmunoassay. Genomic DNA methylation was determined by a Liquid Chromatograph/Mass Spectrometry method, which enables to measure the absolute amount of 5-methylcytosine in DNA. RESULTS: The CEE phase showed significantly decreased plasma tHcy levels compared with placebo phase [14.17 µmol /L (13.28-15.08) vs. 18.71 (16.77-20.86), p<0.05] and significantly increased genomic DNA methylation status in peripheral mononuclear cells (2.85±0.54 ng methylcytosine / µg DNA vs. 2.40±0.43, p<0.05). On the other hand serum folate and B12, and plasma PLP did not show any significant differences between the two phases. CONCLUSIONS: This study demonstrates that estrogen therapy increases genomic DNA methylation status through decreasing tHcy, but not through an alteration of B vitamins status. Since genomic DNA hypomethylation has been widely described in many cancers and pre-cancer tissues, and since DNA methylation is related to gene expression and chromosomal structure, the results of present study suggest that alterations in DNA methylation due to estrogen therapy might be one of the mechanisms for the estrogen related carcinogenesis. This study also suggests that genomic DNA methylation can be a sensitive biomarker of estrogen related carcinogenesis.
Paper presented at the International Symposium on Predictive Oncology and Intervention Strategies; Nice, France; February 7 - 10, 2004; in oral session 898 (Oncogenesis).