Published OnlineFirst December 26, 2014; DOI: 10.1158/1055-9965.EPI-14-0262 Research Article Cancer Epidemiology, Biomarkers Enterolignan-Producing Phenotypes Are & Prevention Associated with Increased Gut Microbial Diversity and Altered Composition in Premenopausal Women in the United States Meredith A.J. Hullar1, Samuel M. Lancaster1,2, Fei Li2, Elizabeth Tseng2, Karlyn Beer2, Charlotte Atkinson3, Kristiina Wah€ al€ a€4, Wade K. Copeland1, Timothy W. Randolph1, Katherine M. Newton5, and Johanna W. Lampe1,2 Abstract Background: Lignans in plant foods are metabolized by gut ysis and regression were used to model the association between bacteria to the enterolignans, enterodiol (END) and enterolac- enterolignan excretion and the GMC. Bacteria associated with tone (ENL). Enterolignans have biologic activities important to ENL production were identified using univariate analysis and the prevention of cancer and chronic diseases. We examined the ridge regression. composition of the gut microbial community (GMC) as a con- Results: After adjusting for dietary fiber intake and adiposity, tributor to human enterolignan exposure. we found a significant positive association between ENL excretion Methods: We evaluated the association between the GMC in and either the GMC (P ¼ 0.0007), or the diversity of the GMC (P ¼ stool, urinary enterolignan excretion, and diet from a 3-day food 0.01). The GMC associated with high ENL production was distinct record in 115 premenopausal (ages 40–45 years) women in the (UNIFRAC, P < 0.003, MRPP) and enriched in Moryella spp., United States. Urinary enterolignans were measured using gas Acetanaerobacterium spp., Fastidiosipila spp., and Streptobacillus spp. chromatography–mass spectroscopy. The GMC was evaluated Conclusion: Diversity and composition of the GMC are asso- using 454 pyrosequencing of the 16S rRNA gene. Sequences were ciated with increased human exposure to enterolignans. aligned in SILVA (www.arb-silva.de). Operational taxonomic Impact: Differences in gut microbial diversity and composition units were identified at 97% sequence similarity. Taxonomic explain variation in gut metabolic processes that affect environ- classification was performed and alpha and beta diversity in mental exposures and influence human health. Cancer Epidemiol relationship to ENL production were assessed. Multivariate anal- Biomarkers Prev; 24(3); 546–54. Ó2014 AACR. Introduction enzymes involved in hormone metabolism, and antitumor activ- ities (10). High interindividual variation in excretion, circulating Epidemiologic studies have shown that the consumption of concentrations, and extent of metabolism of enterolignans exists foods of plant origin is associated with lower risk of several cancers (11). Dietary factors account for a modest amount of the variation (1). In particular, the intake of lignans, which are polyphenolic in enterolignan excretion; and often unaccounted for sources of compounds concentrated in woody portions of plants, seed coats, variation include gastrointestinal transit time, sex, and the com- and the bran layer of grains, has been inversely associated with risk position of the gut microbiome (12, 13). We hypothesize that of breast (2–7) and colon cancer (8, 9). Lignans are converted by variation in the composition of the microbiome influences the the gut microbiota to enterolignans, which are bioactive chemi- exposure of the host to lignan metabolites and that this may cals found in measurable quantities in plasma and urine. Evidence ultimately influence health outcomes. from in vitro and in vivo studies suggests that enterolignans possess Several biochemical steps are required to transform plant a variety of biologic activities relevant to human health, including lignans into enterolignans and each step is likely catalyzed by weak estrogenic and antiestrogenic properties, inhibition of consortia of bacteria that share metabolic intermediates (14). To date, no one bacteria has been identified that can completely 1Fred Hutchinson Cancer Research Center, Seattle, Washington. 2Uni- metabolize the plant lignan, secoisolarisiresinol diglucoside versity of Washington, Seattle, Washington. 3University of Bristol, (SDG) to enterolactone (ENL). For example, isolated Eggerthella Bristol, United Kingdom. 4University of Helsinki, Helsinki, Finland. lenta cannot reduce SECO; however, it can dehydroxylate 2, 3-bis- 5 Group Health Research Institute, Seattle, Washington. (3, 4-dihydroxy-benzyl) butane-1, 4-diol to enterodiol (END), Note: Supplementary data for this article are available at Cancer Epidemiology, one of the intermediary steps in ENL production (12, 15). END Biomarkers & Prevention Online (http://cebp.aacrjournals.org/). can then be converted to ENL by different bacteria (16, 17). Corresponding Author: Meredith A.J. Hullar, Fred Hutchinson Cancer Research Several more bacterial groups likely play similarly unique and Center, Division of Public Health Sciences, Cancer Prevention Program, 1100 complex biochemical roles in the transformation of plant lignans Fairview Avenue North, M4-B402, PO Box 19024, Seattle, WA 98109-1024. to enterolignans (16). Hence, the complexity and diversity Phone: 206-667-1967; Fax: 206-667-7850; E-mail: [email protected] of the gut microbial community (GMC) are essential for maxi- doi: 10.1158/1055-9965.EPI-14-0262 mizing conversion of plant lignans into enterolignans and likely Ó2014 American Association for Cancer Research. influences human exposure to these bacterial compounds. The 546 Cancer Epidemiol Biomarkers Prev; 24(3) March 2015 Downloaded from cebp.aacrjournals.org on October 1, 2021. © 2015 American Association for Cancer Research. Published OnlineFirst December 26, 2014; DOI: 10.1158/1055-9965.EPI-14-0262 Enterolignans Associated with Increased Bacterial Diversity objective of this study was to evaluate the association between Gut microbial community analysis GMC and urinary enterolignan excretion in a well-characterized DNA extraction. DNA was extracted from stool that had been group of premenopausal women. stored in RNAlater at À80C (20). The 16S rRNA gene was amplified and sequenced using 454 pyrosequencing primers Materials and Methods 27f and 519r (V1-V3; ref. 22) for amplicon pyrosequencing (bTE- – Research design and study participants FAP; refs. 23 27) at Research and Testing using Roche 454 FLX This observational study was conducted in premenopausal titanium instruments and reagents and following the manufac- women who were part of a larger study designed to evaluate the turer's guidelines. Sequences have been deposited in the Sequence relationship between bacterial metabolic phenotypes, diet, and Read Archive of NCBI under accession number SRP028900. biomarkers of sex steroid hormone status (18). Of the 203 women in the parent study, 120 collected a fecal sample. Of the 120 16S rRNA gene sequencing and curation. Sequences were compiled women that donated fecal samples, 116 filled out a 3-day food and processed using MOTHUR (v.1.28.0; ref. 28). Sequences were fi record (3DFR) and 101 of the samples were taken within 1 month converted to standard FASTA format from .sff les. Sequences < > of the 3DFR. One woman who provided a stool sample and 3DFR were removed if they were 300 bp, had homopolymers 8 bp, did not have an ENL measurement. Although all participants were more than one mismatch to the forward primer, more than one premenopausal, with normal menstrual cycles, we did not collect mismatch to the barcode, or ambiguous bases. Sequences were urines in conjunction with time in menstrual cycle. Previous work denoised (29), and aligned to the Silva 16S rRNA gene reference by Lampe and colleagues (19) showed no difference in END or alignment (www.arb-silva.de) using the NAST algorithm ENL by phase of cycle in a carefully controlled study of flaxseed (28, 30, 31). Sequences that did not align to the appropriate supplementation. The aims of this study are addressed in this 16S rRNA gene region were removed. Low abundance sequences subset of women. The women were recruited from Group Health, were merged to the high abundant sequences using the pre.cluster a large integrated health plan in Western Washington, and were option in MOTHUR to minimize the effect of pyrosequencing eligible to participate if they were 40 to 45 years and had under- errors in overestimating microbial diversity (32). Potentially gone a screening mammogram in the last 10 months (18). chimeric sequences were removed using ChimeraSlayer (33, 34). Women were excluded if they had more than one prescription for hormone therapy (i.e., oral contraceptives) within 18 months Analysis of the microbiome. Sequences were clustered into oper- of the sampling date; had any history of breast cancer; had breast ational taxonomic units (OTU) at 97% similarity based on the implants; had a hysterectomy or oophorectomy; used tamoxifen average neighbor-joining algorithm. The sequences were classi- fi fi or raloxifene; had any diagnosis of gastrointestinal disorders or ed using the naive Bayesian Classi er trained against an RDP fi gastrointestinal surgeries 10 years before their mammogram; or if training set as implemented in MOTHUR (27). Classi ed they had prescriptions for antibiotics, bisphosphonates, or corti- sequences were assigned to phylum and genus-level phylotypes costeroids within 3 months of their sampling date. All study (35) to characterize the community structure. To characterize the fi parameters were approved by the FHCRC and Group Health IRB alpha diversity, we used OTUs rare ed to 1,265 sequences