Background: Global loss of methylated cytosines in DNA, thought to predispose to chromosomal instability and aneuploidy, has been associated with an increased risk of colorectal neoplasia. Little is known about the relationships between global hypomethylation and lifestyle, demographics, dietary measures, and genetic factors. Methods: Our data were collected as part of a randomized clinical trial testing the efficacy of aspirin and folic acid for the prevention of colorectal adenomas. At a surveillance colonoscopy f3 years after the qualifying exam, we obtained two biopsies of the normal-appearing mucosa from the right colon and two biopsies from the left colon. Specimens were assayed for global hypomethylation using a pyrosequencing assay for LINE-1 (long interspersed nucleotide elements) repeats. Results: The analysis included data from 388 subjects. There was relatively little variability in LINE meth-

Gene-specific promoter methylation of several genes occurs in aging normal tissues and may predispose to tumorigenesis. In the present study, we investigate the association of blood folate levels and dietary and lifestyle factors with CpG island (CGI) methylation in normal colorectal mucosa. Subjects were enrolled in a multicenter chemoprevention trial of aspirin or folic acid for the prevention of large bowel adenomas. We collected 1,000 biopsy specimens from 389 patients, 501 samples from the right colon and 499 from the rectum at the follow-up colonoscopy. We measured DNA methylation of estrogen receptor alpha (ERa) and secreted frizzled related protein-1 (SFRP1), using bisulfite pyrosequencing. We used generalized estimating equations regression analysis to examine the association between methylation and selected variables. For both ERa and SFRP1, percentage methylation was significantly higher in the rectum than in the right colon (P ¼ 0.001). For each 10 years of age, we observed a 1.7% increase in methylation level for ERa and a 2.9% increase for SFRP1 (P < 0.0001). African Americans had a significantly lower level of ERa and SFRP1 methylation than Caucasians and Hispanics. Higher RBC folate levels were associated with higher levels of both ERa (P ¼ 0.03) and SFRP1 methylation (P ¼ 0.01). Our results suggest that CGI methylation in normal colorectal mucosa is related to advancing age, race, rectal location, and RBC folate levels. These data have important implications regarding the safety of supplementary folate administration in healthy adults, given the hypothesis that methylation in normal mucosa may predispose to colorectal neoplasia. Cancer Prev Res; 3(12); 1552-64. Ó2010 AACR.