Unknown,Transcriptomics,Genomics,Proteomics

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Effects of gut-derived trimethylamines on an in vitro model of the blood–brain barrier

ABSTRACT: The experiment was designed to look at the effect (on gene expression) of exposing human cerebromicrovascular endothelial cell line hCMEC/D3 to trimethylamine (0.4 micromolar) or trimethylamine N-oxide (40 micromolar) for 24 h, to determine if circulating metabolites have the potential to affect integrity of the blood-brain barrier.

ORGANISM(S): Homo sapiens

SUBMITTER: Lesley Hoyles

PROVIDER: E-MTAB-6662 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:The incidence of obesity and its related morbidities has rapidly increased over the past twenty years. One consequence of this is the increased probability of having to treat obese individuals for breast cancer. An important co-morbidity associated with obesity are the spectrum of fatty liver diseases. As such, an important question is how treatment of breast cancer with standard therapeutic agents may alter in individuals with fatty liver disease. Such impacts could result in an enhanced adverse effect profile over both acute (e.g. increased cytotoxicity against non-malignant tissues) or chronic (e.g. increased progression through the fatty liver disease spectrum) time periods. In this work we examine if lipid loading in human hepatocytes, leading to a steatotic phenotype, results in an altered toxic liability for the cancer therapeutic doxorubicin. We demonstrate that there is a synergistic impact on cytotoxicity, due to increased oxidative stress. Array analysis reveals the cellular changes associated with hepatocyte adaptation to lipid loading or doxorubicin treatment alone, and how these profiles are significantly altered during doxorubicin treatment of lipid-loaded hepatocytes. Such alterations, we believe, not only underlie the enhanced sensitivity of obese individuals to the acute adverse effects of doxorubicin, but may also potentiate transition along the fatty liver disease spectrum.Experimental design: Huh7 cells were exposed to lipid and/or doxorubicin as required. Huh7 cells were treated with vehicle control or 300 μM FFA mixture for 24 h in T25 culture flasks. The following day, the cells were treated with either 300 μM FFA mixture, 0.1 μM or 3.6 μM DOX and incubated for 4 hrs or 12 hrs. By the end of incubation time, Total RNA was extracted from cells using the RNeasy Plus Mini Kit (QIAGEN-UK) according to the instructions of manufacturer. RNA samples were sent to the Central Biotechnology Services (Cardiff University, UK), for quality control, synthesis of biotin-labeled cRNA, and hybridisation against Illumina Human-HT12 (Illumina, Inc., Hayward, CA) chips. Washed chips were scanned with Bead Station 500x (Illumina) and the signal intensities quantified with BeadStudio (Illumina). To ensure high quality results from the microarray data, RNA samples were extracted from three independent treatments, giving a biological n = 3 per treatment which gives statistical power to increase the confidence of the results/conclusions generated from the microarray experiment.

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Effects of gut-derived trimethylamines on an in vitro model of the blood–brain barrier

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