ORIGINAL ARTICLE Phosphatidylserine Metabolism in Human Lymphoblastic Cells Exposed to Chromium (VI) Angela Gambelunghe, MD, Sandra Buratta, PhD, Giuseppina Ferrara, PhD Student, Rita Mozzi, PhD, Cristina Marchetti, PhD, Nicola Murgia, MD, and Giacomo Muzi, MD Objective: Hexavalent chromium (Cr(VI)) compounds are widely found in different working environments. These compounds can cause apoptosis in hu- man cells, but the mechanisms underlying chromium-induced apoptosis are not clear. A marker of apoptosis is the exposure of phosphatidylserine on cell membrane and the modification of phosphatidylserine metabolism. The aim of this study was to verify whether chromium could cause phosphatidylser- ine exposure and modification of its metabolism in human lymphoblastic leukemia cell line (MOLT-4). Methods: Phosphatidylserine exposure was evaluated by annexin V binding whereas phosphatidylserine metabolism was studied measuring the incorporation of [ 3 H]serine. Results: Cell treatment with Cr(VI) increases phosphatidylserine exposure and cell apoptosis, but decreases the incorporation of [ 3 H]serine into phosphatidylserine in a dose- and time-dependent manner. Conclusions: The Cr(VI)-induced apoptosis also through modification of phosphatidylserine exposure and metabolism. C hromium exists in a series of oxidation states and it is generally accepted that the trivalent (Cr[III]) and hexavalent (Cr[VI]) compounds are the most biologically relevant. Chromium and its compounds are widely used in industry and many workers should be occupationally exposed. Carcinogenicity of chromium, which is used in metallurgical and chemical industries, was first identified over a century ago and Cr(VI) compounds were among the earliest chemicals to be classified as carcinogens. 1 Various studies have been devoted to clarify the mechanism(s) underlying metal toxicity. Hexavalent Chromium uses the anion transport system to enter the cell. Once transported through the cell membrane, Cr(VI) is rapidly reduced to Cr(V) and Cr(IV). These in- termediate states of Cr are reactive and can produce reactive oxygen species (ROS), which cause DNA strand breaks, base modification, lipid peroxidation, and transcription factor activation. In particular, attention has been mainly focused on the generation of ROS and ni- trogen species and on the activation of redox sensitive transcription factors AP-1 and NF-kb, which suggests an effect of this metal on cell signaling. 2 Little information exist on the effects that this metal may have on membrane phospholipids, despite the involvement of phospholipid peroxidation in cell damage 3 and the well known role of membrane phospholipids in cell signaling. Phospholipids participate to cell signaling by various mech- anisms. Glycerophospholipids represent a source of fatty acids From the Department of Experimental and Clinical Medicine, Section of Occupational Medicine, University of Perugia, Piazzale G. Menghini 3 (Drs Gambelunghe, Murgia, and Muzi), Perugia, Italy; Department of Internal Medicine, Section of Biochemistry, University of Perugia, Via del Giochetto (Dr Buratta, Ms Ferrara, and Dr Mozzi), Perugia, Italy; and Department of Experimental and Clinical Medicine, Section of Pharmacology, Toxicology and Chemiotherapy, University of Perugia, Via del Giochetto (Dr Marchetti), Perugia, Italy. This work was financially supported by INAIL (Italian Workers’ Compensation Authority). Address correspondence to: Dr Sandra Buratta, PhD, Department of Internal Medicine, Section of Biochemistry, University of Perugia, Via del Giochetto, Perugia, Italy; E-mail: sandra.buratta@unipg.it. Copyright C  2011 by American College of Occupational and Environmental Medicine DOI: 10.1097/JOM.0b013e31821f2aaf utilized for the synthesis of prostaglandins, leucothrienes, and throm- boxanes and other lipid mediators are produced from membrane sphyngolipids. In addition, phosphatidylserine (PS), which has a mi- nor role in the production of lipid mediators, permits the binding to the membrane of various proteins involved in signal transduction, including protein kinase C. 4 The effect of various metals on the synthesis of lipids in THP-1 cells, a human acute monocytic leukemia cell line, has been studied measuring the incorporation of radioactive acetate in neutral lipids and in glycerophospholipids. 5 This study, which does not report data on PS, demonstrates that chromium, titanium, and cobalt have the same effect of stimulation on the synthesis of triglycerides, but spe- cific different effects on the synthesis of phospholipids. In particular, chromium decreases the synthesis of phosphatidylcholine (PC), tita- nium decreases the synthesis of PC and phosphatidylethanolamine (PE), whereas no effects are observed treating cells with cobalt. Authors suggest that the increase in the synthesis of triglycerides and the decrease in the synthesis of PE and/or PC may be corre- lated. In fact, the last step of the synthesis of these phospholipids and of triglycerides requires the utilization of diglycerides produced from phosphatidic acid. Thus, the increased synthesis of triglyc- erides may be due to the inhibition of ethanolamine and/or choline phosphotrasferase. Phosphatidylserine, which does not represent a significant source of lipid mediators, with the exception of lysoPS, 6 is involved not only in signal transduction, as earlier-mentioned but also in cell apoptosis. In particular, phosphatidylserine is strictly localized on the cytoplasmic side of the plasma membranes and is exposed to the exterior of the cell in particular conditions, such as apopto- sis. This allows recognition and engulfment of apoptotic cells by macrophages. Phosphatidylserine exposure is generally attributed to the activities of flippase and scramblase 7,8 but other mechanisms may be involved, depending on the cell type and/or the apoptotic stimu- lus. In particular, in various cell types subjected to different apop- totic stimuli, PS exposure is accompanied by the stimulation of PS synthesis. 9–11 Some peculiarities of PS metabolism and the lack of stated information on the enzymes responsible for its synthesis 12 make it difficult to establish whether a correlation exists between variation in PS synthesis and physiopathological cell conditions. As opposed to other phospholipids, PS is not synthesized de novo but by a base exchange between the nitrogen base present in membrane PE or PC and free serine. Once synthesized, PS can be decarboxylated into PE, which in turn can be methylated to PC. The relative importance of PS decarboxylation in the production of PE appears to depend on the cell type. Although it is generally accepted that PE is mainly synthesized de novo, PS decarboxyla- tion produces more than 80% of cellular PE in Chinese hamster ovary cells (CHO). 13 The complete conversion PS-PE-PC may be relevant only in particular cell types, such as in neurons where this pathway may produce in loco choline necessary for the synthesis of acetylcholine. 14 Another peculiarity of PS metabolism concerns its degradation by phospholipases that produce lysophospholipids. In fact, the existence of phospholipase A specific for PS and secreted by the cell has been described in mast cells. 15 Copyright © 2011 Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited. 776 JOEM  Volume 53, Number 7, July 2011