DNA methylome-wide alterations associated with estrogen receptor-dependent effects of bisphenols in breast cancer.

BACKGROUND: Bisphenol A (BPA), an estrogen-like endocrine disruptor used in plastics, has been associated with development and promotion of breast cancer, so plastic manufacturers shifted towards less-studied analogs, BPF and BPS. Studying the associated DNA methylome-wide mechanisms of these derivatives is timely, particularly in comparison with BPA. METHODS: We assessed proliferation, cell cycle, and migration of breast cancer cells (estrogen receptor (ER)-positive: MCF-7 and ER-negative: MDA-MB-231) treated with BPF and BPS ± estrogen receptor inhibitor (ERI) in comparison to BPA ± ERI. RNA expression and activity of DNA (de)methylation enzymes and LINE-1 methylation were quantified. DNA methylome-wide analysis was evaluated in bisphenol-exposed cells and compared to clinical breast cancer data. RESULTS: The three bisphenols caused ER-dependent increased proliferation and migration of MCF-7 but not MDA-MB-231 cells, with BPS being 10 times less potent than BPA and BPF. Although they have similar chemical structures, the three bisphenols induced differential DNA methylation alterations at several genomic clusters of or single CpG sites, with the majority of these being ER-dependent. At equipotent doses, BPA had the strongest effect on the methylome, followed by BPS then BPF. No pathways were enriched for BPF while BPA- and BPS-induced methylome alterations were enriched in focal adhesion, cGMP-PKG, and cancer pathways, which were also dysregulated in methylome-wide alterations comparing ER-positive breast cancer samples to adjacent normal tissues. CONCLUSIONS: The three bisphenols have important epigenetic effects in breast cell lines, with those of BPA and BPS overlapping with cancer-related pathways in clinical breast cancer models. Hence, further investigation of their safety is warranted.

BPA exposure is associated with non-monotonic alteration in ESR1 promoter methylation in peripheral blood of men and shorter relative telomere length in peripheral blood of women.

The aim of this study was to evaluate the potential association of urinary Bisphenol A (BPA) levels with estrogen receptor alpha (ESR1) promoter % methylation and relative telomere length in a sample of 482 participants. Urinary BPA concentration was measured using organic phase extraction followed by high performance liquid chromatography mass spectroscopy. Peripheral blood ESR1 promoter % methylation and relative telomere length were measured using direct bisulfite sequencing and real-time polymerase chain reaction, respectively. The mean ± SD urinary BPA concentration adjusted for urinary creatinine was 2.90 ± 4.81 (µg/g creatinine) with a median of 1.86 µg/g creatinine (min-max:

Tamoxifen metabolism predicts drug concentrations and outcome in premenopausal patients with early breast cancer.

Tamoxifen is the standard-of-care treatment for estrogen receptor-positive premenopausal breast cancer. We examined tamoxifen metabolism via blood metabolite concentrations and germline variations of CYP3A5, CYP2C9, CYP2C19 and CYP2D6 in 587 premenopausal patients (Asians, Middle Eastern Arabs, Caucasian-UK; median age 39 years) and clinical outcome in 306 patients. N-desmethyltamoxifen (DM-Tam)/(Z)-endoxifen and CYP2D6 phenotype significantly correlated across ethnicities (R(2): 53%, P<10(-77)). CYP2C19 and CYP2C9 correlated with norendoxifen and (Z)-4-hydroxytamoxifen concentrations, respectively (P<0.001). DM-Tam was influenced by body mass index (P<0.001). Improved distant relapse-free survival (DRFS) was associated with decreasing DM-Tam/(Z)-endoxifen (P=0.036) and increasing CYP2D6 activity score (hazard ratio (HR)=0.62; 95% confidence interval (CI), 0.43-0.91; P=0.013). Low (<14 nM) compared with high (>35 nM) endoxifen concentrations were associated with shorter DRFS (univariate P=0.03; multivariate HR=1.94; 95% CI, 1.04-4.14; P=0.064). Our data indicate that endoxifen formation in premenopausal women depends on CYP2D6 irrespective of ethnicity. Low endoxifen concentration/formation and decreased CYP2D6 activity predict shorter DRFS.

Effects of CYP2B6 genetic polymorphisms in patients receiving cyclophosphamide combination chemotherapy for breast cancer.

PURPOSE: The purpose of this study was to measure the frequency of three CYP2B6 [CYP2B6*4 (rs2279343), CYP2B6*5 (rs3211371) and CYP2B6*9 (rs3745274)] alleles in patients with breast cancer receiving cyclophosphamide (CP) therapy and test whether these variants are predictors of CP-associated toxicity and efficacy. METHODS: A total of 145 female breast cancer patients admitted to the American University of Beirut Medical Center for breast cancer-related therapy were included. Chart review was performed for collection of toxicity data. A time-to-event analysis was performed with a subset of 38 patients. RESULTS: The minor allele frequencies of CYP2B6*9, CYP2B6*4 and CYP2B6*5 were 0.27, 0.29 and 0.07, respectively. CYP2B6 *5/*6, *6/*9 or *6/*6 haplotypes were associated with a significantly shorter time to recurrence of the disease. There were no significant associations with myelo-toxicity. CONCLUSIONS: This is the first report on the pharmacogenetic profile of patients with breast cancer and the therapeutic and myelo-toxic behavior of CP in women from an Arab Middle Eastern country. Our results show that genotyping for these CYP2B6 alleles does not help in personalizing therapy from a toxicity perspective, and the association of shorter survival in these subjects with homozygous variants is interesting yet insufficient to justify routine genotyping prior to therapy, or to consider using a higher CP dose. Larger future studies or meta-analyses will be needed to further clarify the potential implication of these genetic polymorphisms.

Using team-based learning to teach clinical pharmacology in medical school: student satisfaction and improved performance.

Formal teaching in clinical pharmacology was never part of the curriculum at the American University of Beirut Faculty of Medicine. Based on feedback from students and on recommendations of academic bodies, we have introduced, since June 2008, twice-monthly "rational prescribing" sessions during the required internal medicine rotation in year 4 of medical school. All sessions were designed according to the innovative Team-based Learning format and concluded by having the students practice prescription writing and personal formulary development based on the World Health Organization criteria. Our 18-month experience showed that students were very satisfied with the course and the teaching approach, and that their performance on prescription writing and formulary development had improved. Although further studies are needed to explore the impact of team-based learning on additional performance measures, we recommend it as an effective alternative for teaching clinical pharmacology in medical schools.

Evaluation of flurbiprofen urinary ratios as in vivo indices for CYP2C9 activity.

AIMS: We investigated flurbiprofen pharmacokinetics in 12 volunteers to develop a phenotypic trait measure that correlates with the fractional clearance to 4'-hydroxyflurbiprofen. The effect of the CYP2C9 inhibitor fluconazole on flurbiprofen metabolism was also evaluated. METHODS: Flurbiprofen pharmacokinetics were evaluated before and after the first and seventh doses of fluconazole. The urinary recovery ratio was calculated as FLRR = 4'-OHF/[4'-OHF + F(tot)] and the urinary metabolic ratio was calculated as FLMR = 4'-OHF/F(tot), where 4'-OHF and F(tot) represent total (conjugated and unconjugated) amounts recovered in urine. RESULTS: There was a statistically significant relationship between the 4'-OHF formation clearance (4OHCLf) and both the 8-h FLRR and the 8-h FLMR with and without administration of fluconazole. The flurbiprofen apparent oral clearance (CL/F) was decreased by 53% [90% confidence interval (CI) -58, -48] and 64% (90% CI -69, -59), respectively, after administration of one and seven doses of fluconazole when compared with administration of flurbiprofen alone; similarly, the 4OHCLf decreased by 69% (90% CI -74, -64) and 78% (90% CI -83, -73), the 8-h FLRR decreased by 35% (90% CI -41, -29) and 40% (90% CI -46, -35) and the 8-h FLMR decreased by 61% (90% CI -65, -58) and 67% (90% CI -70, -63). The magnitude of decrease in CL/F and 4OHCLf was greater after seven doses compared with after one dose of fluconazole (P < 0.005). CONCLUSIONS: This study provides strong evidence that both the 8-h FLRR and the 8-h FLMR are suitable phenotypic indices for CYP2C9 activity.