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Simultaneously discovering the fate and biochemical effects of pharmaceuticals through untargeted metabolomics

ABSTRACT: Untargeted metabolomics is an established approach in toxicology for characterising endogenous metabolic responses to xenobiotic exposure. Detecting the xenobiotic and its biotransformation products as part of the metabolomics analysis provides an opportunity to simultaneously gain deep insights into its fate and metabolism, and to associate the xenobiotic’s internal relative dose directly with endogenous metabolic responses. This integration of untargeted exposure and response measurements into a single assay has yet to be fully demonstrated. Here we assembled a workflow to discover and analyse xenobiotic-related measurements from routine untargeted UHPLC-MS metabolomics datasets, derived from in vivo (rat plasma and cardiac tissue, and human plasma) and in vitro (human cardiomyocytes) studies that were principally designed to investigate endogenous metabolic responses to drug exposure. Our findings clearly demonstrate how untargeted metabolomics can discover extensive xenobiotic biotransformation maps, temporally-changing relative systemic exposure, and direct associations of endogenous biochemical responses to the internal dose.

INSTRUMENT(S): Liquid Chromatography MS - negative - reverse phase, Liquid Chromatography MS – negative - hilic, Liquid Chromatography MS – positive - reverse phase, Liquid Chromatography MS - positive - hilic, Liquid Chromatography MS - negative - hilic

SUBMITTER: Tara Bowen Ralf Weber

PROVIDER: MTBLS2746 | MetaboLights | 2023-07-03

REPOSITORIES: MetaboLights

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			Action	DRS
	MTBLS2746	Other
	FILES	Other
	a_MTBLS2746_LC-MS_CM_neg_RP_metabolite_profiling.txt	Txt
	a_MTBLS2746_LC-MS_CM_neg_hilic_metabolite_profiling.txt	Txt
	a_MTBLS2746_LC-MS_CM_pos_RP_metabolite_profiling.txt	Txt

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Project description:Animals have developed extensive mechanisms of response to xenobiotic chemical attacks. Although recent genome surveys have suggested a broad conservation of the chemical defensome across metazoans, global gene expression responses to xenobiotics are not known in most invertebrates. Here, using high density tiling arrays with over 2 million probes, we explored genome-wide gene expression in the tunicate Oikopleura dioica in response to two model xenobiotic chemicals – the carcinogenic polycyclic aromatic hydrocarbon benzo[a]pyrene (BaP) the pharmaceutical compound Clofibrate (Clo). The genotoxic compound BaP induced xenobiotic biotransformation and oxidative stress responsive genes, as in vertebrates. Notable exceptions were genes of the aryl hydrocarbon receptor (AhR) signaling pathway. Clo also affected the expression of many biotransformation genes and markedly repressed genes involved in energy metabolism and muscle contraction pathways. Oikopleura appears to have basic defensome toolkit consisting of phase I, phase II and phase III biotransformation genes. Exposure of about 130 four-days-old animals in 1L seawater in glass beakers. Pooled animals used, no replicates. DMSO Treatments: -Clofibrate (Clo) (Sigma-Aldrich, St. Louis, MO): 1 µM and 5 µM. -Benzo[a]pyrene (BaP (Sigma-Aldrich): 0.2 µM and 1 µM - Controls received 1 ml Dimethyl sulfoxide (DMSO). -The animals kept at room temperature and harvested after 10 hrs and frozen. -Total RNA was isolated from the pooled animals using the RNeasy Mini Kit according to manufacturer’s protocols (QIAGEN, Hilden, Germany). - 5 µg total RNA was converted to dscDNA using SuperScript Double-Stranded cDNA Synthesis Kit (Invitrogen, Carlsbad, CA), and dscDNA samples were submitted for microarray analysis. BaP and Clo treated samples were labeled using Cy3-coupled random nonamers and DMSO controls were labeled using Cy5-coupled random nonamers. Total 4 hybridizations for the 8 samples (single hybridization for each treatment and DMSO control pair), no replicates.

Project description:Background: The effects of dose-rate and its implications on radiation biodosimetry methods are not well studied in the context of large-scale radiological scenarios. There are significant health risks to individuals exposed to an acute dose in such an event, but the most realistic scenario would be a combination of exposure to both high and low dose-rates, from both external and internal radioactivity. It is important therefore, to understand the biological response to prolonged exposure; and further, discover biomarkers that can be used to estimate the extent of damage from low-dose rate exposure and propose appropriate clinical treatment. Methods: We irradiated human whole blood ex vivo to three doses, 0.56 Gy, 2.25 Gy and 4.45 Gy, using two dose rates: 1.1Gy/min and 3.1mGy/min. After 24 hours, we isolated RNA from blood cells and hybridized these to Agilent Whole Human genome microarrays. We validated the microarray results using qRT-PCR. Results: Microarray results showed that there were 454 significantly differentially expressed genes after prolonged exposure to all doses. After acute exposure, 598 genes were differentially expressed to all doses combined. Gene ontology terms enriched in both sets of genes were related to immune processes and B cell mediated immunity. Genes responding to acute exposure was also enriched in functions related to natural killer cell activation and cell-to-cell signaling. As expected, p53 pathway was found to be significantly enriched at all doses and by both dose-rates of radiation. Prediction algorithms were able to distinguish between low dose-rate and acute exposures, on the basis of a group of genes. These maybe candidates for preliminary testing as markers for differences in gene expression based on dose-rate. Radiation induced gene expression was measured in ex vivo irradiated human blood, at the 24hr time point after irradiation. Doses (0.56 Gy, 2.2 Gy and 4.45 Gy) were delivered by two dose rates, acute dose rate of 1Gy/min and low dose rate of 3.1 mGy/min.

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Simultaneously discovering the fate and biochemical effects of pharmaceuticals through untargeted metabolomics

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