Perfluorinated alkyl substances in Canadian human milk as part of the Maternal-Infant Research on Environmental Chemicals (MIREC) study.

Perfluorinated alkyl substances (PFAS) were determined in human milk samples (n = 664) from participants in the Maternal-Infant Research on Environmental Chemicals (MIREC) study. ΣPFAS concentrations (sum of seven PFAS) ranged from 3.1 ng L-1 to 603 ng L-1, with a median concentration of 106 ng L-1 in the Canadian mothers' milk analyzed. These data comprise the first pan-Canadian dataset of PFAS in human milk. Perfluorooctanoic acid (PFOA) and linear perfluorooctanesulfonate (L-PFOS) were the dominant contributors to ΣPFAS in human milk samples. An inverse relationship between ΣPFAS concentrations and age was observed (Spearman correlation - 0.184). Primiparous women had elevated PFAS concentrations in milk relative to women who had children previously (p < 0.001). In contrast, the region of maternal birth did not influence ΣPFAS concentrations (p = 0.156). Although China and Norway have observed consistently detectable levels of perfluoroundecanoic acid (PFUdA) in human milk, PFAS with long carbon chains (n ≥ 11) were not present above method detection limits in Canadian human milk samples analyzed as part of the MIREC study. In conclusion, despite the presence of low levels of environmental contaminants in human milk, Health Canada supports breastfeeding due to the benefits to both infants and mothers.

A phosphorylation-and-ubiquitylation circuitry driving ATR activation and homologous recombination.

RPA-coated single-stranded DNA (RPA-ssDNA), a nucleoprotein structure induced by DNA damage, promotes ATR activation and homologous recombination (HR). RPA is hyper-phosphorylated and ubiquitylated after DNA damage. The ubiquitylation of RPA by PRP19 and RFWD3 facilitates ATR activation and HR, but how it is stimulated by DNA damage is still unclear. Here, we show that RFWD3 binds RPA constitutively, whereas PRP19 recognizes RPA after DNA damage. The recruitment of PRP19 by RPA depends on PIKK-mediated RPA phosphorylation and a positively charged pocket in PRP19. An RPA32 mutant lacking phosphorylation sites fails to recruit PRP19 and support RPA ubiquitylation. PRP19 mutants unable to bind RPA or lacking ubiquitin ligase activity also fail to support RPA ubiquitylation and HR. These results suggest that RPA phosphorylation enhances the recruitment of PRP19 to RPA-ssDNA and stimulates RPA ubiquitylation through a process requiring both PRP19 and RFWD3, thereby triggering a phosphorylation-ubiquitylation circuitry that promotes ATR activation and HR.

Levels and temporal trend of bisphenol A in composite food samples from Canadian Total Diet Study 2008-2012.

Food composite samples from the Canadian Total Diet Study which was conducted each year from 2008 to 2012 rotating between different cities were analysed for bisphenol A (BPA). The overall levels of BPA in the composite food samples from each of the five years from 2008 to 2012 were similar in general with averages (range) of 7.7 ng/g (0.20-106 ng/g), 7.8 ng/g (0.26-110 ng/g), 6.9 ng/g (0.20-84 ng/g), 7.7 ng/g (0.20-105 ng/g) and 9.0 ng/g (0.15-90 ng/g) for 2008, 2009, 2010, 2011 and 2012, respectively. Levels of BPA in most of the non-canned food composite samples were low and no particular trends were observed. In contrast, the trend of BPA levels in canned food composite samples over the five years (2008-2012) varies. BPA levels in most of the canned food composite samples from 2008 to 2012 were consistent in general (e.g. canned luncheon meat: 10-18 ng/g, canned baked beans: 18-25 ng/g). While BPA levels over the five years were found to decrease for some canned food composite samples (e.g., canned fish: 109 ng/g in 2009 vs. 51 ng/g in 2012), they were also found to increase for some other canned food composite samples (e.g. canned meat soups: 90-104 ng/g in 2011-2012 vs. 29 ng/g in 2008). Thus, recent changes in can coating for food packaging to BPA-free alternatives may have not been fully reflected in all canned food products over the period from 2008 to 2012. Continued monitoring is necessary to more fully assess the potential impact on dietary exposure by the use of BPA alternatives in food contact materials.

The homeobox gene EMX2 is a prognostic and predictive marker in malignant pleural mesothelioma.

OBJECTIVES: Malignant pleural mesothelioma (MPM) is a highly aggressive neoplasm with a poor prognosis and limited treatment options. EMX2 is a homeobox transcription factor that may regulate key developmental pathways known to promote tumorigenesis. In this study, we evaluated the prognostic and predictive significance of EMX2 expression in MPM. MATERIALS AND METHODS: Fifty surgically resected MPM specimens were studied. Quantitative real-time RT-PCR was used to analyze EMX2 mRNA expression. Association of EMX2 levels with clinical outcomes was evaluated with using the Kaplan-Meier method and a multivariate Cox proportional hazards regression model. RESULTS: EMX2 expression was significantly associated with IMIG stage (p<0.001) and smoking history (p=0.006). Cox hazard regression modeling identified low-EMX2 expression as a negative prognostic factor in progression-free survival by both univariate (p=0.002) and multivariate analysis (p=0.002). Kaplan-Meier analysis revealed significant differences in progression-free survival between low- and high-EMX expressing groups in all patients (p=0.001), and also when grouped by early (I/II) stage disease (p<0.001), patients undergoing pleurectomy (p<0.001) and patients with an epitheliod subtype (p<0.004). Furthermore, EMX2 expression predicted response to neoadjuvant chemotherapy. High-EMX2 expression was associated with decreased progression-free survival after neoadjuvant therapy, suggesting that induction therapy should be avoided in these patients. CONCLUSIONS: EMX2 expression is downregulated in advanced cases of malignant pleural mesothelioma and may serve as an important prognostic and predictive molecular biomarker of progression-free survival.

Down-regulation of SIX3 is associated with clinical outcome in lung adenocarcinoma.

BACKGROUND: Lung cancer is a common cancer and the leading cause of cancer-related death worldwide. SIX3 is a human homologue of the highly conserved sine oculis gene family essential during embryonic development in vertebrates, and encodes a homeo-domain containing transcription factor. Little is known about the role of SIX3 in human tumorigenesis. This study is to assess the expression/function of SIX3 and the significance of SIX3 as a prognostic biomarker in lung adenocarcinoma. METHODS: Quantitative real-time RT-PCR was used to analyze SIX3 mRNA expression and quantitative methylation specific PCR (MSP) was used to examine promoter methylation. MTS and colony formation assays were performed to examine cell proliferation. Wound healing assays were used to assess cell migration, and microarrays were utilized to examine genes regulated by SIX3 in lung cancer cells. Association of SIX3 expression levels with clinical outcomes of patients with lung adenocarcinoma was evaluated using the Kaplan-Meier method and a multivariate Cox proportional hazards regression model. RESULTS: SIX3 was down-regulated in lung adenocarcinoma tissues compared to their matched adjacent normal tissues, and this down-regulation was associated with methylation of the SIX3 promoter. SIX3 was also methylation-silenced in lung cancer cell lines. Restoration of SIX3 in lung cancer cells lacking endogenous SIX3 suppressed cell proliferation and migration, and downregulated a number of genes involved in proliferation and metastasis such as S100P, TGFB3, GINS3 and BAG1. Moreover, SIX3 mRNA expression was associated with significantly improved overall survival (OS) and progression-free survival (PFS) in adenocarcinoma patients and patients with bronchioloalveolar carcinoma (BAC) features. CONCLUSIONS: SIX3 may play an important role as a novel suppressor in human lung cancer. SIX3 has potential as a novel prognostic biomarker for patients with lung adenocarcinomas.

Bisphenol a in baby food products in glass jars with metal lids from Canadian markets.

A method based on solid phase extraction and derivatization with acetic anhydride followed by gas chromatography-mass spectrometry was validated for the determination of bisphenol A (BPA) in baby foods. The average method detection limit (MDL) was 0.18 ng/g for a 5 g sample. Method repeatability was demonstrated with the replicate analyses of various different types of baby foods; relative standard deviations (RSD) ranged from 1.2 to 16.1% with an average of 8.7%. Extraction recoveries ranged from 93.5 to 102.5% for different types of baby foods spiked at levels of 1-8 ng/g. This method was used to analyze 122 baby food products of 7 brands in glass jars with metal lids for BPA. The presence of BPA could not be confirmed and quantified for 23 of the 122 products due to interference from sample matrices. For the other 99 products, 15% had BPA levels of less than the average MDL, about 70% had BPA levels of less than 1 ng/g, and the average BPA levels in all 99 products was 1.1 ng/g. The average BPA level in the baby food products from brand E (3.9 ng/g) is higher than the average BPA levels in the products from the other brands (0.54-1.1 ng/g). The highest level of BPA, 7.2 ng/g, was found in two products from brand E as well. The average BPA level in the fruit products from all brands (0.60 ng/g) is lower than those in the mixed-dish products (1.1 ng/g) and the vegetable products (1.2 ng/g).

Levels of bisphenol A diglycidyl ether (BADGE) and bisphenol F diglycidyl ether (BFDGE) in canned liquid infant formula products in Canada and dietary intake estimates.

A method based on solid-phase extraction followed by HPLC analysis with fluorescence detection was developed for the determination of bisphenol A diglycidyl ether (BADGE) and bisphenol F diglycidyl ether (BFDGE) in liquid infant formula. In this method, instead of trying to isolate and measure each individual form of the molecules, hydrolysis of BADGE, BFDGE, BADGE x H2O, and BFDGE x H2O was forced to completion to their stable forms, BADGE x 2H2O and BFDGE x 2H2O, before extraction. The method LODs were 2.0 ng/g for BADGE and 3.0 ng/g for BFDGE. Extraction recoveries were 61-91% for BADGE, and 55-82% for BFDGE over the concentration range of 10 to 50 ng/g. The method was used to analyze samples of 21 canned liquid infant formula products for BADGE and BFDGE. BADGE was detected in samples of all products at levels ranging from as low as 2.4 ng/g to as high as 262 ng/g. BFDGE was detected in only one product (40 ng/g), and this product also had the highest BADGE level (262 ng/g). HPLC/MS/MS with a similar LOD was also used to confirm the results. The probable daily intakes (PDI) of BADGE and BFDGE due to consumption of canned liquid infant formula were estimated for infants from premature to 12-18 months of age. The maximum PDI of BADGE was 22 microg/kg body weight/day for the 12-18 months old with the maximum formula intake. The maximum PDI of BFDGE was < 3.4 microg/kg body weight/day.

Inhibition of Hsp90 leads to cell cycle arrest and apoptosis in human malignant pleural mesothelioma.

INTRODUCTION: Heat shock protein 90 (Hsp90) is an abundant molecular chaperone that mediates the maturation and stability of a variety of proteins associated with the promotion of cell growth and survival. Inhibition of Hsp90 function leads to proteasomal degradation of its mis-folded client proteins. Recently, Hsp90 has emerged as being of prime importance to the growth and survival of cancer cells and its inhibitors have already been used in phase I and II clinical trials. METHODS: We investigated how 17-allylamino-17-demethoxygeldanamycin (17-AAG), a small molecule inhibitor of Hsp90, is implicated in human malignant pleural mesothelioma (MM). RESULTS: We found that 17-AAG led to significant G1 or G2/M cell cycle arrest, inhibition of cell proliferation, and decrease of AKT, AKT1, and survivin expression in all human malignant pleural mesothelioma cell lines examined. We also observed significant apoptosis induction in all MM cell lines treated with 17-AAG. Furthermore, 17-AAG induced apoptosis in freshly cultured primary MM cells and caused signaling changes identical to those in 17-AAG treated MM cell lines. CONCLUSION: These results suggest that Hsp90 is strongly associated with the growth and survival of MM and that inhibition of Hsp90 may have therapeutic potential in the treatment of MM.

Levels of bisphenol A in canned liquid infant formula products in Canada and dietary intake estimates.

A sensitive, efficient, and reproducible method, based on solid phase extraction and derivatization with acetic anhydride followed by gas chromatography-mass spectrometry in selected-ion monitoring mode, was developed for the determination of bisphenol A (BPA) in liquid infant formula. The method quantification limit was 0.5 ng g(-1). Extraction recoveries were 85-94% over the concentration range of 2.5-20 ng g(-1). Good reproducibility of the method was observed at levels of 0.54 and 10.4 ng g(-1) with relative standard deviations of 5.0 and 2.8%, respectively. The method was used to analyze samples of 21 canned liquid infant formula products for BPA. BPA was detected in all samples at levels ranging from as low as 2.27 ng g(-1) to as high as 10.2 ng g(-1). The probable daily intakes of BPA due to consumption of canned liquid infant formula were estimated for infants from premature to 12-18 months of age. The maximum probable daily intake of BPA was 1.35 microg kg(-1) of body weight day(-1) for 0-1-month-old infants with the maximum formula intake, which is below the provisional tolerable daily intake for BPA established by Health Canada, 25 microg kg(-1) of body weight day(-1).

Epigenetic alteration of the Wnt inhibitory factor-1 promoter occurs early in the carcinogenesis of Barrett's esophagus.

The role of Wnt antagonists in the carcinogenesis of esophageal adenocarcinoma (EAC) remains unclear. We hypothesized that downregulation of the Wnt inhibitory factor-1 (WIF-1) might be involved in the neoplastic progression of Barrett's esophagus (BE). We analyzed the DNA methylation status of the WIF-1 promoter in normal, preneoplastic, and neoplastic samples from BE patients and in EAC cell lines. We investigated the role of WIF-1 on EAC cell growth and the chemosensitization of the cells to cisplatin. We found that silencing of WIF-1 correlated with promoter hypermethylation. EAC tissue samples showed higher levels of WIF-1 methylation compared to the matched normal epithelium. In addition, we found that WIF-1 hypermethylation was more frequent in BE samples from patients with EAC than in BE samples from patients who had not progressed to EAC. Restoration of WIF-1 in cell lines where WIF-1 was methylation-silenced resulted in growth suppression. Restoration of WIF-1 could sensitize the EAC cells to the chemotherapy drug cisplatin. Our results suggest that silencing of WIF-1 through promoter hypermethylation is an early and common event in the carcinogenesis of BE. Restoring functional WIF-1 might be used as a new targeted therapy for the treatment of this malignancy.

Inhibition of Wnt-2 and galectin-3 synergistically destabilizes beta-catenin and induces apoptosis in human colorectal cancer cells.

Constitutive activation of the Wnt pathway as a result of APC, AXIN1 or CTNNB1 mutations has been found in most colorectal cancers. For a long time, this aberrant Wnt activation has been thought to be independent of upstream signals. However, recent studies indicate that upstream signals retain their ability to regulate the Wnt pathway even in the presence of downstream mutations. Wnt-2 is well known for its overexpression in colorectal cancer. Galectin-3 (Gal-3), a multifunctional carbohydrate binding protein implicated in a variety of biological functions, has recently been reported to interact with beta-catenin. In this study, we investigated roles of Wnt-2 and Gal-3 in the regulation of canonical Wnt/beta-catenin signaling. We found that siRNA silencing of either Wnt-2 or Gal-3 expression inhibited TCF-reporter activity, decreased cytosolic beta-catenin level and induced apoptosis in human colorectal cancer cells containing downstream mutations. More interestingly, we showed that inhibition of both Wnt-2 and Gal-3 had synergistic effects on suppressing canonical Wnt signaling and inducing apoptosis, suggesting that aberrant canonical Wnt/beta-catenin signaling in colorectal cancer can be regulated at multiple levels. The combined inhibition of Wnt-2 and Gal-3 may be of superior therapeutic advantage to inhibition by either one of them, giving rise to a potential development of novel drugs for the targeted treatment of colorectal cancer.

Wnt inhibitory factor-1 gene transfer inhibits melanoma cell growth.

Silencing of Wnt antagonists with aberrant activation of Wnt signaling is a common phenomenon in various human cancers. Wnt inhibitory factor-1 (WIF-1) is a secreted antagonist of Wnt signaling and acts through direct binding to Wnt in the extracellular space. In this study, we tried to illuminate the impact of WIF-1 gene expression in melanoma with WIF-1 silencing by in vitro and in vivo studies. We restored the expression of WIF-1 by nonviral gene transfer with a pcDNA3.1 vector. We demonstrated inhibition of melanoma cell growth after WIF-1 restoration in colony formation and proliferation assays in vitro. In addition, the inhibitory effect was related to downregulation of Wnt signaling, which was demonstrated at both the transcriptional and translational levels. Furthermore, by using a xenograft mouse model, we confirmed the effect of WIF-1 expression in suppressing tumor growth by inhibition of Wnt signaling in vivo. Our results suggest the potential for further application of WIF-1 gene therapy in melanoma with WIF-1 silencing.

Targeting the Wnt signaling pathway to treat Barrett's esophagus.

Barrett's esophagus (BE) is an acquired condition in which the normal squamous epithelium in the distal esophagus is replaced by a metaplastic columnar epithelium, as a complication of chronic gastroesophageal reflux. The clinical significance of this disease is its associated predisposition to esophageal adenocarcinoma (EAC). Recently, and similarly to other human malignancies, the Wnt signaling pathway and its key component beta-catenin have been implicated in the carcinogenesis of BE. Although mutations in adenomatous polyposis coli (APC) or beta-catenin are rare in EAC, alterations of upstream components, such as overexpression of Wnt2 ligand or downregulation of Wnt antagonists may play dominant roles in the activation of the Wnt pathway. Increasing evidence suggests that inhibiting the Wnt pathway may be a new targeted therapy for the treatment of cancers and could, therefore, be promising for the cure of EAC, which remains a highly lethal disease.

Wnt inhibitory factor-1, a Wnt antagonist, is silenced by promoter hypermethylation in malignant pleural mesothelioma.

Wnt inhibitory factor-1 (WIF-1) is a secreted protein that antagonizes Wnt signaling. We recently demonstrated the importance of aberrant activation of the Wnt signaling pathway in various cancers including malignant pleural mesothelioma. In this study, we revealed downregulated WIF-1 expression in cell lines and primary tissue when compared to normal mesothelial cell lines and adjacent pleura, respectively. We observed hypermethylation in four of four mesothelioma cell lines, but not in two normal mesothelial cell lines. In primary tissue samples, we observed methylation in three paired tumor specimens compared to their adjacent normal pleura and methylation in eight of nine unpaired tumor tissue samples. Taken together, our studies suggest that WIF-1 silencing due to its promoter hypermethylation is an important mechanism underlying the constitutively activated Wnt signaling in mesothelioma. New therapies toward inhibition of the Wnt pathway through WIF-1 might be promising for the future treatment of malignant mesothelioma.

Methylation of APC, TIMP3, and TERT: a new predictive marker to distinguish Barrett's oesophagus patients at risk for malignant transformation.

Barrett's associated oesophageal adenocarcinoma (EAC) is one of the most rapidly increasing malignancies in Western countries. Because of its poor prognosis, management of this disease through screening of Barrett's oesophagus (BE) patients and identification of those with a high risk of developing an adenocarcinoma seems a promising approach. Early molecular markers of malignant transformation might contribute to such screening approaches. Gene promoter methylation analysis was performed on normal, pre-neoplastic, and neoplastic lesions from BE patients. All lesions of interest were sampled by microdissection from formalin-fixed paraffin-embedded tissue sections. We found that, in 27 adenocarcinomas, APC, TIMP3, TERT, CDKN2A, and SFRP1 promoters were methylated in 93%, 65%, 64%, 48%, and 91%, respectively; in contrast MLH1, RASSF1, RARB, CDH1, and FHIT promoters were methylated in less than 5% of the tumours. In BE mucosa from patients who had progressed to adenocarcinoma (12 samples), APC, TIMP3, and TERT promoters were hypermethylated in 100%, 91%, and 92% of cases, whereas in BE mucosa from patients who had not progressed (16 samples) methylation was found only in 36%, 23%, and 17%, respectively. Furthermore, the epigenetic profile of BE with and without EAC differed significantly with, respectively, 81% and 26% of the PCR samples showing promoter hypermethylation for APC, TIMP3, and TERT (p < 0.0001). Promoter methylation of CDKN2A was infrequently detected in BE samples, while SFRP1 methylation was observed in all samples. Our results suggest that promoter methylation profiling of BE using multiple target genes including APC, TIMP3, and TERT might be used as a predictive marker for increased EAC risk.

Monoallelic methylation of the APC promoter is altered in normal gastric mucosa associated with neoplastic lesions.

Adenomatous polyposis coli (APC) promoter hypermethylation has been reported frequently in normal gastric mucosa, but it remained to be clarified whether this occurs in every individual. In this study, methylation of the APC promoter was analyzed in histologically normal-appearing gastric mucosa samples by methylation-sensitive single-strand conformation analysis and by a methylation-sensitive dot blot assay. Epithelial cell samples were collected by microdissection from tissue sections. Equal amounts of methylated and unmethylated APC alleles were found in all gastric mucosa samples from patients without any gastric lesions (20 samples). Allele-specific methylation analysis showed that the methylation of the APC promoter was monoallelic; however, which allele was methylated depended on the cell type. Increased or decreased methylation was found in 10 of 36 (28%) normal gastric mucosa samples adjacent to a gastric or esophageal adenocarcinoma. No allelic loss was found at the APC locus. Modification of the methylation status was also found in 3 of 21 (14%) normal-appearing gastric mucosa samples adjacent to intestinal metaplasia. In contrast, all normal mucosa samples in cases with chronic gastritis but without metaplasia or dysplasia showed a monoallelic methylation pattern. Our results indicate the following: (a) In normal gastric mucosa, the APC promoter shows monoallelic methylation, which is not due to imprinting but most likely due to allelic exclusion; (b) the excluded allele differs between foveolar and glandular epithelial cells; (c) the APC methylation pattern is frequently altered in normal-appearing gastric mucosa of gastric or esophageal adenocarcinoma patients; and (d) such alterations also occur in normal gastric mucosa adjacent to intestinal metaplasia.

Methylation-sensitive single-strand conformation analysis: a rapid method to screen for and analyze DNA methylation.

The last few years have seen a growing interest in the study of DNA methylation because of its now acknowledged implication in cancer. The use of bisulfite to convert unmethylated cytosine to uracil, even as methylated cytosine remains unchanged, constitutes the basis for differentiating between methylated and unmethylated specific CpG sites in CpG islands. This technique therefore is critical to the success of this approach. Different parameters have to be considered in order to achieve a total conversion of cytosines to uracils. Several bisulfite-based methods are available for analyzing DNA methylation status. Methylation-sensitive single-strand conformation analysis (MS-SSCA) yields specific and semiquantitative data. The method is based on bisulfite treatment of DNA followed by polymerase chain reaction using primers without a CpG site to avoid selective amplification of either methylated or unmethylated DNA, and finally by single-strand conformation analysis (SSCA). The method allows one to establish clonal variations in the DNA methylation status for clones representing as little as 5-10% of the total cell population. MS-SSCA has, furthermore, a broad application field since it is the appropriate method for the analysis of frozen, fixed, and even microdissected tissues.

Peptabodies as tools to test ligands isolated from phage-displayed peptide libraries.

We have previously isolated filamentous bacteriophages, expressing linear hexa-peptides and homing to bone marrow endothelium (BME), by panning in vivo of a phage-displayed peptide library in mice. Here, we used peptabody fusion proteins to test the binding capacity of the hexa-peptide SSLTTG to BME cells in vitro. To display this motif in a multimeric form, as originally presented on the bacteriophage, we expressed it N-terminally as a fusion with the peptabody cartilage oligomeric matrix assembly protein (COMP) pentamerization domain, either alone or followed by the N1 domain of the pIII phage coat protein. Binding of the peptabody constructs to the mouse BME cell line STR-10 was investigated by immunofluorescence using anti-COMP antibodies. Only peptabody fusion proteins co-expressing pIII-N1 exhibited binding to STR-10, regardless of the presence or absence of SSLTTG. These results indicate that the phage coat protein pIII-N1 domain is the principle determinant responsible for the binding of filamentous bacteriophages to cells of the reticulo-endothelial system (RES). Peptabodies expressing pIII-N1 did not bind to the osteoblast-like cell line MC3T3-E1, indicating that binding is mediated by receptors specifically expressed by BME cells in vivo. Polyinosinic acid (poly-I) was able to inhibit binding of bacteriophages and pIII-N1 expressing peptabodies to STR-10, confirming our previous studies showing that bacteriophages bind to scavenger receptors (SR) expressed by BME cells. In summary, the present study shows the usefulness of peptabodies as a general tool to test the binding capacity of peptide ligands identified by phage display.