Characterization of Glucocorticoid Receptor Promoter Methylation in Breast Cancer

Epidemiological studies have identified psychological stress as a significant risk factor in breast cancer. The stress response is regulated by the HPA axis in the brain and is mediated by glucocorticoid receptor (GR) signalling. It has been found that early life events can affect epigenetic programming of GR, and methylation of the GR promoter has been reported in colorectal tumourigenesis. Decreased GR expression has also been observed in breast cancer. In addition, it has been previously demonstrated that unliganded GR can serve as a direct activator of the BRCA1 promoter in mammary epithelial cells. We propose a model whereby methylation of the GR promoter in the breast significantly lowers GR expression, resulting in insufficient BRCA1 promoter activation and an increased risk of developing cancer. Antibody-based methylated DNA enrichment was followed by qPCR analysis (MeDIP-qPCR) in a novel assay developed to detect locus-specific methylation levels. It was found that 13% of primary breast tumours were hypermethylated at the GR proximal promoter whereas no methylation was detected in normal tissue. RT-PCR and 5’ RACE analysis identified exon 1B as the predominant alternative first exon in the breast. Tumours methylated near exon 1B had decreased GR expression compared to unmethylated samples, suggesting that this region is important for transcriptional regulation of GR. It was also determined that GR and BRCA1 expression was decreased in breast tumour compared to normal tissue. Furthermore, the relative expression of GR and BRCA1 measured by qRT-PCR was correlated in normal tissue but this association was not found in tumour tissue. From this, it appears that lower GR levels with associated decreased BRCA1 expression in tissues may be a predisposing factor for breast cancer. Based on these results we propose a role for GR as a potential tumour suppressor gene in the breast due to its association with BRCA1, also a tumour suppressor gene, as well as its consistently decreased expression in breast tumours and methylation of its proximal promoter in a subset of cancer patients.

Laboratory Investigation of Diluted Bitumen Trapping and Dissolution in Gravel

The Canadian economy is largely dependent on the distribution of large volumes of oil to domestic and international markets by a long network of pipelines. Unfortunately, accidents occur, and oil can leak or spill from these pipelines before it reaches its destination. Of particular concern are the long-term consequences of oil spills in freshwater, which include sinking of oil in water and the contamination of sensitive areas, such as where fish (e.g., salmon) deposit their eggs in gravel-dominated river sediments. There is a knowledge gap regarding the fate and behaviour of oil in river sediment. To this end, this study aimed at finding the potential for diluted bitumen (dilbit) oil to become trapped in gravel and to transfer hydrocarbons into water by dissolution, which are harmful to aquatic life. Two sets of laboratory experiments were conducted to simulate conditions of an oil spill on an exposed bank or in shallow water. In the first set, by conducting capillary pressure-saturation (Pc-Sw) experiments it was found that dilbit can enter gravel pores without much resistance and approximately 14% of the pore volume can be occupied by discontinuous single or multipore blobs of dilbit following imbibition of water. Air-water Pc-Sw experiments done in laboratory 1-D columns required gravity correction and could be successfully scaled to predict dilbit-water Pc-Sw curves, except for the trapped amount of dilbit. Trapped dilbit constituents can be dissolved into the water flowing through gravel pores (hyporheic flow) at different velocities. In the second set, dissolution experiments suggested that for the duration of the test, hydrocarbons that cause acute toxicity dissolve rapidly, likely resulting in a decrease in their effective solubility. However, dilbit saturation changed only <2% within that time. Chronically toxic PAH compounds were also detected in the effluent water. The total concentration of all detected PAHs and alkylPAHs exceeded the threshold literature value to protect early-life stage fish. Observations of decreased concentrations with increased aqueous velocities as well as less than equilibrium concentrations indicated that the mass transfer was rate-limited. A correlation was developed for the mass transfer rate coefficient to understand the mass transfer behaviour beyond the conditions used in the experiments, which had a Reynolds number exponent similar to the studies of NAPL dissolution in groundwater.