Caffeine Inhibits EGF-Stimulated Trophoblast Cell Motility through the Inhibition of mTORC2 and Akt.

Impaired trophoblast invasion is associated with pregnancy disorders such as early pregnancy loss and preeclampsia. There is evidence to suggest that the consumption of caffeine during pregnancy may increase the risk of pregnancy loss; however, little is known about the direct effect of caffeine on normal trophoblast biology. Our objectives were to examine the effect of caffeine on trophoblast migration and motility after stimulation with epidermal growth factor (EGF) and to investigate the intracellular signaling pathways involved in this process. Primary first-trimester extravillous trophoblasts (EVT) and the EVT-derived cell line SGHPL-4 were used to study the effect of caffeine on EGF-stimulated cellular motility using time-lapse microscopy. SGHPL-4 cells were further used to study the effect of caffeine and cAMP on EGF-stimulated invasion of fibrin gels. The influence of caffeine and cAMP on EGF-stimulated intracellular signaling pathways leading to the activation of Akt were investigated by Western blot analysis. Caffeine inhibits both EGF-stimulated primary EVT and SGHPL-4 cell motility. EGF stimulation activates phosphatidylinositol 3-kinase, and Akt and caffeine inhibit this activation. Although cAMP inhibits both motility and invasion, it does not inhibit the activation of Akt, indicating that the effects of caffeine seen in this study are independent of cAMP. Further investigation indicated a role for mammalian target of rapamycin complex 2 (mTORC2) as a target for the inhibitory effect of caffeine. In conclusion, we demonstrate that caffeine inhibits EGF-stimulated trophoblast invasion and motility in vitro and so could adversely influence trophoblast biology in vivo

A UPLC-MS based exploration of the xenobiotic and endogenous metabolic phenotypes of pre-clinical models of hepatotoxicity

To reduce late stage attrition during drug development, and improve the diagnosis of drug induced liver injury (DILI), a greater mechanistic understanding of DILI and improved predictive biomarkers are required. In this thesis, the xenobiotic and endogenous metabolic phenotypes of model hepatotoxins are studied in the rat using an ultra-performance liquid chromatography- mass spectrometry (UPLC-MS) based metabonomics approach. The idiosyncratic hepatotoxin Tienilic Acid (TA), was compared to its structural analogue, Tienilic Acid Isomer (TAI), which is an intrinsic hepatotoxin. TAI dosing resulted in elevated ALT activity and liver necrosis, whereas TA showed no signs of toxicity. The untargeted UPLC-MS approach revealed both previously reported and novel TA drug metabolites, including likely acyl-glucuronides and amino acid conjugates. In contrast, the TAI metabolites detected were predominantly glutathione (GSH) related; reflective of higher reactive metabolite formation. Untargeted UPLC-MS and targeted ion-pair-LC-MS revealed numerous endogenous metabolic alterations, including an elevation in hepatic and plasma ophthalmic acid, common to TA and TAI treated animals. In addition, a unique elevation in pyroglutamate was detected in response to TAI. Interestingly, both ophthalmic acid and pyroglutamic acid have previously been associated with hepatic GSH depletion and oxidative stress. Hepatic oxidative stress is a well-established mechanism in intrinsic toxicity, but has a less established role in idiosyncratic toxicity. To enable further assessment of these compounds, and other glutathione related metabolites, as potential biomarkers of hepatic oxidative stress, a quantitative UPLC-MS/MS assay was developed. Interestingly, despite TA and TAI both depleting hepatic GSH and elevating ophthalmic acid, they were found to impact other circulating metabolites in different ways. To further explore the dynamics of these metabolites, the assay was applied to plasma from paracetamol (APAP) dosed rats; a model GSH depleting hepatotoxin. Quantitative data such as these may contribute to the further development and validation of mathematical models to predict hepatic glutathione status from multiple circulating plasma biomarkers. This thesis demonstrates the utility of a UPLC-MS based approach for hypothesis generation and biomarker development.Open Acces

Ultrahigh-performance liquid chromatography tandem mass spectrometry with electrospray ionization quantification of tryptophan metabolites and markers of gut health in serum and plasma-application to clinical and epidemiology cohorts

A targeted ultrahigh-performance liquid chromatography tandem mass spectrometry with electrospray ionization (UHPLC-ESI-MS/MS) method has been developed for the quantification of tryptophan and its downstream metabolites from the kynurenine and serotonin pathways. The assay coverage also includes markers of gut health and inflammation, including citrulline and neopterin. The method was designed in 96-well plate format for application in multiday, multiplate clinical and epidemiology population studies. A chromatographic cycle time of 7 min enables the analysis of two 96-well plates in 24 h. To protect chromatographic column lifespan, samples underwent a two-step extraction, using solvent protein precipitation followed by delipidation via solid-phase extraction (SPE). Analytical validation reported accuracy of each analyte &lt;20% for the lowest limit of quantification and &lt;15% for all other quality control (QC) levels. The analytical precision for each analyte was 2.1-12.9%. To test the applicability of the method to multiplate and multiday preparations, a serum pool underwent periodic repeat analysis during a run consisting of 18 plates. The % CV (coefficient of variation) values obtained for each analyte were &lt;15%. Additional biological testing applied the assay to samples collected from healthy control participants and two groups diagnosed with inflammatory bowel disease (IBD) (one group treated with the anti-inflammatory 5-aminosalicylic acid (5-ASA) and one group untreated), with results showing significant differences in the concentrations of picolinic acid, kynurenine, and xanthurenic acid. The short analysis time and 96-well plate format of the assay makes it suitable for high-throughput targeted UHPLC-ESI-MS/MS metabolomic analysis in large-scale clinical and epidemiological population studies.</p