Mammary epithelium permeability during established lactation: associations with cytokine levels in human milk.

Objective: The cytokine profile of human milk may be a key indicator of mammary gland health and has been linked to infant nutrition, growth, and immune system development. The current study examines the extent to which mammary epithelium permeability (MEP) is associated with cytokine profiles during established lactation within a sample of US mothers. Methods: Participants were drawn from a previous study of human milk cytokines. The present analysis includes 162 participants (98 Black, 64 White) with infants ranging from 1 to 18 months of age. Levels of cytokines were determined previously. Here we measure milk sodium (Na) and potassium (K) levels with ion-selective probes. Two approaches were used to define elevated MEP: Na levels ≥10 mmol/L and Na/K ratios greater than 0.6. Associations between maternal-infant characteristics, elevated MEP, and twelve analytes (IL-6, IL-8, TNFα, IL-1ß, FASL, VEGFD, FLT1, bFGF, PLGF, EGF, leptin, adiponectin) were examined using bivariate associations, principal components analysis, and multivariable logistic regression models. Results: Elevated MEP was observed in 12 and 15% of milk samples as defined by Na and Na/K cutoffs, respectively. The odds of experiencing elevated MEP (defined by Na ≥ 10 mmol/L) were higher among Black participants and declined with older infant age. All cytokines, except leptin, were positively correlated with either Na or the Na/K ratio. A pro-inflammatory factor (IL-6, IL-8, TNFα, IL-1ß, EGF) and a tissue remodeling factor (FASL, VEGFD, FLT1, bFGF, PLGF, adiponectin) each contributed uniquely to raising the odds of elevated MEP as defined by either Na or the Na/K ratio. Conclusion: This exploratory analysis of MEP and cytokine levels during established lactation indicates that elevated MEP may be more common in US populations than previously appreciated and that individuals identifying as Black may have increased odds of experiencing elevated MEP based on current definitions. Research aimed at understanding the role of MEP in mammary gland health or infant growth and development should be prioritized.

Rapid cell isolation in breastmilk in a non-clinical setting by a deterministic lateral displacement device and selective water and fat absorption.

Breastmilk is a reliable source of biomarker-containing, sloughed breast cells that have the potential to give valuable health insights to new mothers. Furthermore, known DNA-based markers for pregnancy-associated breast cancer are chemically stable and can be safely stored on a commercially available FTA® Elute Micro (EM) card, which can subsequently be mailed to a testing facility for the cost of a stamp. In theory, this archiving process can be performed by nonprofessionals in very low-resource settings as it simply requires placing a drop of breastmilk on an EM card. Although this level of convenience is paramount for new mothers, the low cell density of breastmilk complicates archiving on an EM card as such commercial products and associated protocols were designed for high-cell density physiological fluids such as blood. In this study, we present the use of a deterministic lateral displacement (DLD) device combined with porous superabsorbent polymers and hydrophobic sponges to achieve simple and low-cost cell enrichment in breastmilk. As the critical separation diameter in a DLD device is more heavily dependent on lithographically controlled pillar layout than fluid or flow properties, our use of DLD microfluidics allowed for the accommodation of both varying viscosities in human breastmilk samples and a varying pressure of actuation resulting from manual, syringe-driven operation. We demonstrate successful cell enrichment (>11×) and a corresponding increase in the DNA concentration of EM card elutions among breastmilk samples processed with our hybrid microfluidic system. As our device achieves sufficiently high cell enrichment in breastmilk samples while only requiring the user to push a syringe for 4 min with reasonable effort, we believe that it has high potential to expand EM card DNA archiving for diagnostic applications with low-cell density physiological fluids and in low-resource settings.

Prediagnostic White Blood Cell DNA Methylation and Risk of Breast Cancer in the Prostate Lung, Colorectal, and Ovarian Cancer Screening Trial (PLCO) Cohort.

BACKGROUND: White blood cell (WBC) DNA may contain methylation patterns that are associated with subsequent breast cancer risk. Using a high-throughput array and samples collected, on average, 1.3 years prior to diagnosis, a case-cohort analysis nested in the prospective Sister Study identified 250 individual CpG sites that were differentially methylated between breast cancer cases and noncases. We examined five of the top 40 CpG sites in a case-control study nested in the Prostate, Lung, Colorectal, and Ovarian Cancer Screening Trial (PLCO) Cohort. METHODS: We investigated the associations between prediagnostic WBC DNA methylation in 297 breast cancer cases and 297 frequency-matched controls. Two WBC DNA specimens from each participant were used: a proximate sample collected 1 to 2.9 years and a distant sample collected 4.2-7.3 years prior to diagnosis in cases or the comparable timepoints in controls. WBC DNA methylation level was measured using targeted bisulfite amplification sequencing. We used logistic regression to obtain ORs and 95% confidence intervals (CI). RESULTS: A one-unit increase in percent methylation in ERCC1 in proximate WBC DNA was associated with increased breast cancer risk (adjusted OR = 1.29; 95% CI, 1.06-1.57). However, a one-unit increase in percent methylation in ERCC1 in distant WBC DNA was inversely associated with breast cancer risk (adjusted OR = 0.83; 95% CI, 0.69-0.98). None of the other ORs met the threshold for statistical significance. CONCLUSIONS: There was no convincing pattern between percent methylation in the five CpG sites and breast cancer risk. IMPACT: The link between prediagnostic WBC DNA methylation marks and breast cancer, if any, is poorly understood.

Inter-Individual Variation in Response to Estrogen in Human Breast Explants.

Exposure to estrogen is strongly associated with increased breast cancer risk. While all women are exposed to estrogen, only 12% are expected to develop breast cancer during their lifetime. These women may be more sensitive to estrogen, as rodent models have demonstrated variability in estrogen sensitivity. Our objective was to determine individual variation in expression of estrogen receptor (ER) and estrogen-induced responses in the normal human breast. Human breast tissue from female donors undergoing reduction mammoplasty surgery were collected for microarray analysis of ER expression. To examine estrogen-induced responses, breast tissue from 23 female donors were cultured ex- vivo in basal or 10 nM 17ß-estradiol (E2) media for 4 days. Expression of ER genes (ESR1 and ESR2) increased significantly with age. E2 induced consistent increases in global gene transcription, but expression of target genes AREG, PGR, and TGFß2 increased significantly only in explants from nulliparous women. E2-treatment did not induce consistent changes in proliferation or radiation induced apoptosis. Responses to estrogen are highly variable among women and not associated with levels of ER expression, suggesting differences in intracellular signaling among individuals. The differences in sensitivity to E2-stimulated responses may contribute to variation in risk of breast cancer.

Prediagnostic breast milk DNA methylation alterations in women who develop breast cancer.

Prior candidate gene studies have shown tumor suppressor DNA methylation in breast milk related with history of breast biopsy, an established risk factor for breast cancer. To further establish the utility of breast milk as a tissue-specific biospecimen for investigations of breast carcinogenesis, we measured genome-wide DNA methylation in breast milk from women with and without a diagnosis of breast cancer in two independent cohorts. DNA methylation was assessed using Illumina HumanMethylation450k in 87 breast milk samples. Through an epigenome-wide association study we explored CpG sites associated with a breast cancer diagnosis in the prospectively collected milk samples from the breast that would develop cancer compared with women without a diagnosis of breast cancer using linear mixed effects models adjusted for history of breast biopsy, age, RefFreeCellMix cell estimates, time of delivery, array chip and subject as random effect. We identified 58 differentially methylated CpG sites associated with a subsequent breast cancer diagnosis (q-value <0.05). Nearly all CpG sites associated with a breast cancer diagnosis were hypomethylated in cases compared with controls and were enriched for CpG islands. In addition, inferred repeat element methylation was lower in breast milk DNA from cases compared to controls, and cases exhibited increased estimated epigenetic mitotic tick rate as well as DNA methylation age compared with controls. Breast milk has utility as a biospecimen for prospective assessment of disease risk, for understanding the underlying molecular basis of breast cancer risk factors and improving primary and secondary prevention of breast cancer.

Differences in Genome-wide DNA Methylation Profiles in Breast Milk by Race and Lactation Duration.

Black women in the United States are disproportionately affected by early-onset, triple-negative breast cancer. DNA methylation has shown differences by race in healthy and tumor breast tissues. We examined associations between genome-wide DNA methylation levels in breast milk and breast cancer risk factors, including race, to explain how this reproductive stage influences a woman's risk for, and potentially contributes to racial disparities in, breast cancer. Breast milk samples and demographic, behavioral, and reproductive data, were obtained from cancer-free, uniparous, and lactating U.S. black (n = 57) and white (n = 82) women, ages 19-44. Genome-wide DNA methylation analysis was performed on extracted breast milk DNA using the Infinium HumanMethylation450 BeadChip. Statistically significant associations between breast cancer risk factors and DNA methylation beta values, adjusting for potential confounders, were determined using linear regression followed by Bonferroni Correction (P < 1.63 × 10-7). Epigenetic analysis in breast milk revealed statistically significant associations with race and lactation duration. Of the 284 CpG sites associated with race, 242 were hypermethylated in black women. All 227 CpG sites associated with lactation duration were hypomethylated in women who lactated longer. Ingenuity Pathway Analysis of differentially methylated promoter region CpGs by race and lactation duration revealed enrichment for networks implicated in carcinogenesis. Associations between DNA methylation and lactation duration may offer insight on its role in lowering breast cancer risk. Epigenetic associations with race may mediate social, behavioral, or other factors related to breast cancer and may provide insight into potential mechanisms underlying racial disparities in breast cancer incidence.

Dietary Intervention to Increase Fruit and Vegetable Consumption in Breastfeeding Women: A Pilot Randomized Trial Measuring Inflammatory Markers in Breast Milk.

BACKGROUND: Diets rich in fruits and vegetables (F/V) can reduce the inflammatory profile of circulating cytokines and potentially decrease the risk of breast cancer. However, the extent to which a diet rich in F/V alters cytokine levels in breast tissue remains largely unknown. Breast milk provides a means of assessing concentrations of secreted cytokines in the breast microenvironment and is a potential tool for studying the effects of diet on inflammation in breast tissue and breast cancer risk. OBJECTIVE: The aim of this pilot randomized trial was to test the feasibility of increasing F/V intake in breastfeeding women and of measuring changes in markers of inflammation in breast milk. DESIGN AND INTERVENTION: Participants randomized to the intervention (n=5) were provided weekly boxes of F/V, along with dietary counseling, to increase consumption of F/V to 8 to 10 daily servings for 12 consecutive weeks. Controls (n=5) were directed to the US Department of Agriculture's "ChooseMyPlate" diet for pregnancy and breastfeeding. PARTICIPANTS/SETTING: Ten breastfeeding women consuming fewer than five servings of F/V per day, as estimated by the National Institutes of Health "All-Day" Fruit and Vegetable Screener (F/V Screener), were recruited through flyers and a lactation consultant between February and May 2016 in the Western Massachusetts area. MAIN OUTCOME MEASURES: Baseline demographic and F/V intake data were collected during enrollment. At week 1 and week 13 (final) home visits, participants provided milk samples and anthropometric measurements were recorded. Participants completed F/V screeners at baseline and at study end. Adiponectin, leptin, C-reactive protein, and 11 additional cytokines were measured in breast milk collected at weeks 1 and 13. STATISTICAL ANALYSES: F/V consumption at baseline and after the final visit, and between controls and intervention groups, was compared with dependent and independent t tests, respectively. Differences between cytokine levels at weeks 1 and 13 were assessed with a mixed-effects repeated-measures model. RESULTS: All women in the intervention increased F/V intake and were consuming more servings than controls by week 13; daily serving of F/V at baseline and final visit: controls=1.6 and 2.0, diet=2.6 and 9.9. Most cytokines were detected in the majority of milk samples: 12 were detected in 90% to 100% of samples, one was detected in 75% of samples, and one was detected in 7.5% of samples; coefficients of variation were below 14% for 11 of the cytokines. CONCLUSIONS: These preliminary findings indicate that it is feasible to significantly increase F/V intake in breastfeeding women and provide support for conducting a larger diet intervention study in breastfeeding women, in which longer-term benefits of the intervention are assessed.

Pro-inflammatory cytokines and growth factors in human milk: an exploratory analysis of racial differences to inform breast cancer etiology.

BACKGROUND: Analysis of cytokines and growth factors in human milk offers a noninvasive approach for studying the microenvironment of the postpartum breast, which may better reflect tissue levels than testing blood samples. Given that Black women have a higher incidence of early-onset breast cancers than White women, we hypothesized that milk of the former contains higher levels of pro-inflammatory cytokines, adipokines, and growth factors. METHODS: Participants included 130 Black and 162 White women without a history of a breast biopsy who completed a health assessment questionnaire and donated milk for research. Concentrations of 15 analytes in milk were examined using two multiplex and 4 single-analyte electrochemiluminescent sandwich assays to measure pro-inflammatory cytokines, angiogenesis factors, and adipokines. Mixed-effects ordinal logistic regression was used to identify determinants of analyte levels and to compare results by race, with adjustment for confounders. Factor analysis was used to examine covariation among analytes. RESULTS: Thirteen of 15 analytes were detected in ≥ 25% of the human milk specimens. In multivariable models, elevated BMI was significantly associated with increased concentrations of 5 cytokines: IL-1ß, bFGF, FASL, EGF, and leptin (all p-trend < 0.05). Black women had significantly higher levels of leptin and IL-1ß, controlling for BMI. Factor analysis of analyte levels identified two factors related to inflammation and growth factor pathways. CONCLUSION: This exploratory study demonstrated the feasibility of measuring pro-inflammatory cytokines, adipokines, and angiogenesis factors in human milk, and revealed higher levels of some pro-inflammatory factors, as well as increased leptin levels, among Black as compared with White women.

TROP2 methylation and expression in tamoxifen-resistant breast cancer.

BACKGROUND: The DNA methyltransferase 1 inhibitor, 5-Aza-2'-deoxycytidine (5-Aza-dC) is a potential treatment for breast cancer. However, not all breast tumors will respond similarly to treatment with 5-Aza-dC, and little is known regarding the response of hormone-resistant breast cancers to 5-Aza-dC. METHODS: We demonstrate that 5-Aza-dC-treatment has a stronger effect on an estrogen receptor-negative, Tamoxifen-selected cell line, TMX2-28, than on the estrogen receptor-positive, MCF7, parental cell line. Using data obtained from the HM450 Methylation Bead Chip, pyrosequencing, and RT-qPCR, we identified a panel of genes that are silenced by promoter methylation in TMX2-28 and re-expressed after treatment with 5-Aza-dC. RESULTS: One of the genes identified, tumor associated calcium signal transducer 2 (TACSTD2), is altered by DNA methylation, and there is evidence that in some cancers decreased expression may result in greater proliferation. Analysis of DNA methylation of TACSTD2 and protein expression of its product, trophoblast antigen protein 2 (TROP2), was extended to a panel of primary (n = 34) and recurrent (n = 34) breast tumors. Stratifying tumors by both recurrence and ER status showed no significant relationship between TROP2 levels and TACSTD2 methylation. Knocking down TACSTD2 expression in MCF7 increased proliferation however; re-expressing TACSTD2 in TMX2-28 did not inhibit proliferation, indicating that TACSTD2 re-expression alone was insufficient to explain the decreased proliferation observed after treatment with 5-Aza-dC. CONCLUSIONS: These results illustrate the complexity of the TROP2 signaling network. However, TROP2 may be a valid therapeutic target for some cancers. Further studies are needed to identify biomarkers that indicate how TROP2 signaling affects tumor growth and whether targeting TROP2 would be beneficial to the patient.

Potential of breastmilk analysis to inform early events in breast carcinogenesis: rationale and considerations.

This review summarizes methods related to the study of human breastmilk in etiologic and biomarkers research. Despite the importance of reproductive factors in breast carcinogenesis, factors that act early in life are difficult to study because young women rarely require breast imaging or biopsy, and analysis of critical circulating factors (e.g., hormones) is often complicated by the requirement to accurately account for menstrual cycle date. Accordingly, novel approaches are needed to understand how events such as pregnancy, breastfeeding, weaning, and post-weaning breast remodeling influence breast cancer risk. Analysis of breastmilk offers opportunities to understand mechanisms related to carcinogenesis in the breast, and to identify risk markers that may inform efforts to identify high-risk women early in the carcinogenic process. In addition, analysis of breastmilk could have value in early detection or diagnosis of breast cancer. In this article, we describe the potential for using breastmilk to characterize the microenvironment of the lactating breast with the goal of advancing research on risk assessment, prevention, and detection of breast cancer.

ELF5 and DOK7 regulation in anti-estrogen treated cells and tumors.

BACKGROUND: Most women with primary breast cancers that express estrogen receptor alpha (ER or ESR1) are treated with endocrine therapies including the anti-estrogen tamoxifen, but resistance to these anti-endocrine therapies often develops. This study characterizes the expression of hormone receptors, and the mRNA and DNA methylation levels of docking protein 7 (DOK7), and E74-like factor 5 (ELF5), in 21 novel tamoxifen-resistant cell lines and extends the findings to primary and recurrent human breast tumors. METHODS: Twenty-one tamoxifen-selected cell lines were developed through cloning by limiting dilution of an MCF-7 cell culture treated with 1 µM tamoxifen for 6 months. The parent (MCF-7) and tamoxifen-selected cell lines were characterized for protein expression of ER, progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2) using immunohistochemistry (IHC). The mRNA levels of ER, DOK7, and ELF5 were assessed using quantitative RT-PCR. Promoter methylation levels of DOK7 and ELF5 were determined by pyrosequencing of bisulfite-modified DNA. The relationship between hormone receptor status and promoter methylation of DOK7 and ELF5 was further examined using available methylation array data (Illumina HM450) from a set of paired primary and second breast tumors from 24 women. RESULTS: All 21 of the novel tamoxifen-selected cell lines are ER-positive, and HER2-negative, and 18 of the cell lines are PR-negative while the MCF-7 cells were scored as ER-positive, modestly PR-positive and HER2 negative. Expression of DOK7 and ELF5 is significantly up-regulated in half of the tamoxifen-selected cell lines as compared to the parental MCF-7. In contrast, the previously established ER-negative TMX2-28 cell line has decreased expression of both DOK7 and ELF5 and increased DNA methylation in the transcriptional start site region of these genes. ELF5 methylation was lower in second versus primary tumors in women who received anti-estrogen treatment, in PR-negative versus PR-positive tumors, and in the subset of PR-positive first tumors from the group of women who had second PR-negative tumors as compared to those who had second PR-positive tumors. CONCLUSIONS: The distinct ELF5 methylation of PR-positive primary tumors from women who had a PR-negative recurrence indicates the possibility of stratification of women for tailored treatment in the early stages of disease.

Association of TGF-ß2 levels in breast milk with severity of breast biopsy diagnosis.

PURPOSE: TGF-ß plays a dual role in breast carcinogenesis, acting at early stages as tumor-suppressors and later as tumor-promoters. TGF-ß isoforms are expressed in breast tissues and secreted in milk, suggesting that analysis of levels in milk might be informative for breast cancer risk. Accordingly, we assessed TGF-ß2 levels in milk from women who had undergone a breast biopsy and related the concentrations to diagnosis. METHODS: Milk donated by women who had undergone or were scheduled for a breast biopsy was shipped on ice for processing and testing. Breast cancer risk factors were obtained through a self-administered questionnaire, and biopsy diagnoses were extracted from pathology reports. TGF-ß2 levels in milk, assessed as absolute levels and in relation to total protein, were analyzed in bilateral samples donated by 182 women. Linear regression was used to estimate relationships of log-transformed TGF-ß2 levels and TGF-ß2/ total protein ratios to biopsy category. RESULTS: Milk TGF-ß2 levels from biopsied and non-biopsied breasts within women were highly correlated (r (2) = 0.77). Higher mean TGF-ß2 milk levels (based on average of bilateral samples) were marginally associated with more severe breast pathological diagnosis, after adjusting for duration of nursing current child (adjusted p trend = 0.07). CONCLUSIONS: Our exploratory analysis suggests a borderline significant association between higher mean TGF-ß2 levels in breast milk and more severe pathologic diagnoses. Further analysis of TGF-ß signaling in milk may increase understanding of postpartum remodeling and advance efforts to analyze milk as a means of assessing risk of breast pathology.

Promoter methylation in epithelial-enriched and epithelial-depleted cell populations isolated from breast milk.

BACKGROUND: Breast cancer is the most frequently diagnosed cancer among Turkish women and both the incidence and associated mortality appear to be increasing. Of particular concern is the percentage of young women diagnosed with breast cancer; roughly 20% of all breast cancer diagnoses in Turkey are in women younger than 40 years. Increased DNA methylation in the promoter region of tumor suppressor genes is a promising molecular biomarker, and human milk provides exfoliated breast epithelial cells appropriate for DNA methylation analyses. Comparisons between DNA methylation patterns in epithelial (epithelial-enriched) and nonepithelial (epithelial-depleted) cell fractions from breast milk have not been reported previously. OBJECTIVE: In the present study, we examined promoter methylation of 3 tumor suppressor genes in epithelial-enriched and epithelial-depleted cell fractions isolated from breast milk of 43 Turkish women. METHODS: Percentage methylation in the promoter region of Rass association domain family 1 (RASSF1), secreted frizzle related protein 1 (SFRP1), and glutathione-S-transferase class pi 1 was determined by pyrosequencing of the epithelial-enriched and epithelial-depleted cell fractions. RESULTS: Pyrosequencing identified a few subjects with significantly increased methylation in 1 or more genes. There was little correlation between the 2 cell fractions within individuals; only 1 woman had increased methylation for 1 gene (SFRP1) in both her enriched and depleted cell fractions. Methylation was positively associated with age for SFRP1 (epithelial-depleted fraction) and with body mass index for RASSF1 (epithelial-enriched cell fraction), respectively. CONCLUSION: Overall, results show that the methylation signals vary between different cell types in breast milk and suggest that breast milk can be used to assess DNA methylation patterns associated with increased breast cancer risk.

Determination of free Bisphenol A (BPA) concentrations in breast milk of U.S. women using a sensitive LC/MS/MS method.

Bisphenol A (BPA) is a synthetic, endocrine-disrupting compound. Free BPA has been detected in human samples indicating that humans are internally exposed to estrogenically active BPA. The purpose of this study was to develop a sensitive method to detect free BPA in human breast milk. BPA was isolated from the milk of 21 nursing mothers in the U.S. by solid-phase extraction. It was then derivatized to improve sensitivity and subsequently analyzed by ultra high performance liquid chromatography-tandem mass spectrometry. Free BPA was detected in 62% of the milk samples (≤ 0.22-10.8 ng mL(-1), median 0.68 ng mL(-1), mean 3.13 ng mL(-1)). No statistical difference in BPA concentrations was observed between women with a low or high Body Mass Index (BMI) (<30 (n=11) and>30 (n=10), respectively). However, there was a significant association between BPA concentration and race. Caucasian women had significantly higher levels of free BPA in their breast milk than non-Caucasian women (mean=4.44 (n=14) and 0.52 (n=7), respectively; p<0.05). The difference between races could be attributed to variations in exposure, lifestyle or metabolism and suggests that certain populations should take extra precautions to limit BPA exposure, particularly during pregnancy and lactation.

Differential expression of cancer-related proteins in paired breast milk samples from women with breast cancer.

BACKGROUND: Breast cancer risk increases during pregnancy and remains elevated for a number of years thereafter. Cancer-associated proteins that are secreted into breast milk may provide a means to detect cancer in the lactating breast or to assess future breast cancer risk. OBJECTIVE: To determine whether proteins linked to breast cancer would be differentially expressed in matched (both breasts from each participant) human milk samples collected from women with unilateral breast cancer. METHODS: Five cancer-associated proteins (basic fibroblast growth factor [bFGF], YKL-40, neutrophil gelatinase-associated lipocalin, and transforming growth factor ß1 and ß2) were analyzed in milk provided by 5 lactating women, 4 of whom were known to have cancer in 1 breast (and the opposite breast clinically disease free) at the time of milk collection and 1 who developed breast cancer 2 years after milk collection. RESULTS: Expression was significantly higher for TGFß2 (P = .03) and bFGF (P =.03) in the breasts with cancer. CONCLUSION: These proteins may play a role in assessing a woman's risk of pregnancy-associated breast cancer. Because of variable protein concentration among patients and the limited sample size, the results are considered preliminary.

Differential expression of cancer associated proteins in breast milk based on age at first full term pregnancy.

BACKGROUND: First full term pregnancy (FFTP) completed at a young age has been linked to low long term breast cancer risk, whereas late FFTP pregnancy age confers high long term risk, compared to nulliparity. Our hypothesis was that proteins linked to breast cancer would be differentially expressed in human milk collected at three time points during lactation based on age at FFTP. METHODS: We analyzed breast milk from 72 lactating women. Samples were collected within 10 days of the onset of lactation (baseline-BL), two months after lactation started and during breast weaning (W). We measured 16 proteins (11 kallikreins (KLKs), basic fibroblast growth factor, YKL-40, neutrophil gelatinase-associated lipocalin and transforming growth factor (TGF) ß-1 and -2) associated with breast cancer, most known to be secreted into milk. RESULTS: During lactation there was a significant change in the expression of 14 proteins in women < 26 years old and 9 proteins in women > = 26 at FFTP. The most significant (p < .001) changes from BL to W in women divided by FFTP age (< 26 vs. > = 26) were in KLK3,6, 8, and TGFß2 in women < 26; and KLK6, 8, and TGFß2 in women > = 26. There was a significant increase (p = .022) in KLK8 expression from BL to W depending on FFTP age. Examination of DNA methylation in the promoter region of KLK6 revealed high levels of methylation that did not explain the observed changes in protein levels. On the other hand, KLK6 and TGFß1 expression were significantly associated (r2 = .43, p = .0050). CONCLUSIONS: The expression profile of milk proteins linked to breast cancer is influenced by age at FFTP. These proteins may play a role in future cancer risk.

Increased promoter methylation in exfoliated breast epithelial cells in women with a previous breast biopsy.

Accurately identifying women at increased risk of developing breast cancer will provide greater opportunity for early detection and prevention. DNA promoter methylation is a promising biomarker for assessing breast cancer risk. Breast milk contains large numbers of exfoliated epithelial cells that are ideal for methylation analyses. Exfoliated epithelial cells were isolated from the milk obtained from each breast of 134 women with a history of a non-proliferative benign breast biopsy (Biopsy Group). Promoter methylation of three tumor suppressor genes, RASSF1, SFRP1 and GSTP1, was assessed by pyrosequencing of bisulfite-modified DNA. Methylation scores from the milk of the 134 women in the Biopsy Group were compared to scores from 102 women for whom a breast biopsy was not a recruitment requirement (Reference Group). Mean methylation scores for RASSF1 and GSTP1 were significantly higher in the Biopsy than in the Reference Group. For all three genes the percentage of outlier scores was greater in the Biopsy than in the Reference Group but reached statistical significance only for GSTP1. A comparison between the biopsied and non-biopsied breasts of the Biopsy Group revealed higher mean methylation and a greater number of outlier scores in the biopsied breast for both SFRP1 and RASSF1, but not for GSTP1. This is the first evidence of CpG island methylation in tumor suppressor genes of women who may be at increased risk of developing breast cancer based on having had a prior breast biopsy.

In vitro hepatic metabolism of 2,2',4,4',5-pentabromodiphenyl ether (BDE 99) in Chinook salmon (Onchorhynchus tshawytscha).

Polybrominated diphenyl ethers (PBDEs) are brominated flame retardants that persist in the environment and are present in geographically widespread fish species. PBDE concentrations can be particularly high in resident Chinook salmon (Onchorhynchus tshawytscha) in the Puget Sound, Washington. Although PBDE residues in salmon and other fish are often dominated by lower brominated congeners, these congeners are not produced commercially in the greatest quantity, suggesting bioaccumulation of the lower molecular weight PBDEs or debromination of more fully brominated congeners. We determined the capacity of Chinook liver fractions to debrominate 2,2',4,4',5-pentabromodiphenyl ether (BDE 99), a model PBDE congener readily debrominated by common carp (Cyprinus caprio). Liver subcellular fractions from two strains of Chinook were incubated with BDE 99 prior to liquid/liquid extraction followed by gas chromatography/mass spectrometry analysis (GC/MS analysis) to identify metabolites and debromination products. In contrast to common carp, debromination of BDE 99 to BDE 47 (2,2',4,4'-tetrabromodiphenyl ether) was not observed in microsomal fractions from either strain of Chinook salmon. However, Chinook salmon liver microsomes from both Chinook strains slowly debrominated BDE 99 to BDE 49 (2,2',4,5'-tetrabromodiphenyl ether), a unique debromination product whose formation has not been reported in other fish. Three-year-old males belonging to a Rapid River Spring Chinook salmon genetic strain showed a somewhat greater microsomal debromination capacity than older hatchery returning male Chinook, but were still inefficient in the debromination of BDE 99 relative to carp. Microsomal debromination of BDE 99 to BDE 49 was not NADPH-dependent, indicating a lack of cytochrome P450 involvement. By contrast, omission of the reductant dithiothreitol (DTT) from Chinook microsomal preparations resulted in a lack of BDE 99 debromination, suggesting the involvement of a microsomal reductase(s) or deiodinase (DI). Cytosolic fractions from Chinook salmon and Common carp debrominated BDE 99 to BDE 49 in vitro. However, carp cytosolic enzymes preferentially formed BDE 47. In summary, our data indicate significant differences among teleosts with respect to efficiency and metabolite profiles of BDE 99 debromination, and suggest that the high concentrations of BDE 47 in resident Chinook salmon from the Puget Sound are not a result of hepatic metabolism of BDE 99. The results of our study also suggest the involvement of an unidentified hepatic reductase or DI in PBDE debromination in fish.