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Excessive accumulation of phospholipids leads to phospholipidosis (PL), which disrupts cellular functions, in extreme cases leading to acute or chronic disease. Citalopram and many other cationic amphiphilic drugs (CADs) have been shown to cause PL both in vitro and in vivo. Recent toxicogenomic studies suggest four hypothetical mechanisms for PL (lysosomal enzyme transport decrease, lysosomal phospholipase activity decrease, phospholipids biosynthesis increase or cholesterol biosynthesis increase). However, the post-genomic steps remain largely unknown and proteomic analyses hold significant promise for defining mechanisms of PL induction. In this study U937 monoblastoid cells were exposed to citalopram hydrobromide for 24 h (0, 20, 100 or 200 microM as citalopram free base) and then harvested for whole cell proteomic analysis using 2-D gel electrophoresis, or transmission electron microscopy (TEM). Protein spots that were significantly altered versus controls were analysed by MALDI-TOF mass spectrometry. Up-regulated proteins were Glyoxalase-I (Glo 1) and 3-Hydroxy-3-methylglutaryl-Coenzyme A synthase 1 (HMGCS1) in cells with PL shown by TEM (favouring the cholesterol biosynthesis increase hypothesis for citalopram induced PL). Other altered proteins were catalase (up-regulated), beta-actin (up-regulated) and 14-3-3 protein (down-regulated). The function of several of the successfully identified proteins indicates a potential perturbed lipid metabolism.

Objective: To investigate the possible mechanisms underlying the liver enzyme elevations seen during clinical studies of long-term treatment (>35 days) with ximelagatran, and investigate the usefulness of pre-clinical in vitro systems to predict drug-induced liver effects., Methods: Ximelagatran and its metabolites were tested for effects on cell viability, mitochondrial function, formation of reactive metabolites and reactive oxygen species, protein binding, and induction of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) gene expression or nuclear orphan receptors. Experimental systems included fresh and cryopreserved hepatocytes, human hepatoma cell lines (HepG2 and HuH-7) and subcellular human liver fractions., Results: Loss of cell viability was only seen in HepG2 cells at ximelagatran concentrations 100 microM and in cryopreserved human hepatocytes at 300 microM, while HuH-7 cells were not affected by 24 h exposure at up to 300 microM ximelagatran. Calcium homeostasis was not affected in HepG2 cells exposed to ximelagatran up to 300 microM for 15 min. There was no evidence for the formation of reactive metabolites when cell systems were exposed to ximelagatran. ALT and AST expression in human hepatoma cell lines were also unchanged by ximelagatran. Mitochondrial functions such as respiration, opening of the transition pore, mitochondrial membrane depolarization and beta-oxidation were not affected by ximelagatran or its metabolites., Conclusion: Ximelagatran at concentrations considerably higher than that found in plasma following therapeutic dosing had little or no effect on cellular functions studied in vitro. The in vitro studies therefore did not elucidate the mechanism by which ximelagatran induces liver effects in humans, possibly because of limitations in the experimental systems not reflecting characteristics of the human hepatocyte, restricted exposure time, or because the primary mechanism for the observed clinical liver effects is not on the parenchymal liver cell.