Changes in protein expression associated with chronic in vitro exposure of hexavalent chromium to osteoblasts and monocytes: A proteomic approach Vijay Krishna Raghunathan, 1 M. Helen Grant, 1 Elizabeth M. Ellis 2 1 Bioengineering Unit, Wolfson Centre, 106 Rottenrow, University of Strathclyde, Glasgow, G4 0NW, United Kingdom 2 Strathclyde Institute of Pharmacy and Biomedical Sciences, University of Strathclyde, Glasgow, G4 0NR, United Kingdom Received 29 August 2008; accepted 20 November 2008 Published online 20 February 2009 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jbm.a.32396 Abstract: Cr (VI) is a well-recognized environmental toxin and carcinogen. It is known to be released from orthopedic metal implants in-situ by biocorrosion and is speculated to play a role in periprosthetic osteolysis. It is hence essential to understand its long-term biological effects. We have assessed the in vitro responses of osteoblasts and monocytes to chronic exposure (3 weeks) to Cr (VI), at concentrations that have been measured in patients with metal implants, using two-dimensional gel electrophoresis. Cr (VI) exposure resulted in a differential time-dependent regulation of gly- colytic, stress, and cytoskeletal proteins. The proteins that have been found to be altered in expression play an essential role in normal cellular functioning such as energy metabo- lism, cell signaling, and proliferation. The results highlight the complex molecular changes that occur in both cell types with long-term exposure to Cr and may be useful in estab- lishing a series of clinically useful biomarkers to monitor long-term use of metallic implants. Ó 2009 Wiley Periodi- cals, Inc. J Biomed Mater Res 92A: 615–625, 2010 Key words: chronic in vitro toxicity; hexavalent chromium; monocytes; osteoblasts; metal orthopaedic implants INTRODUCTION Stainless steel and cobalt-chrome alloys are often used in the manufacture of medical implants for joint replacement. Hip replacement is the second most common elective operation in Europe and often involves the use of metal-on-metal (MOM) articula- tions. MOM implants are associated with high circu- lating levels of metal ions especially cobalt and chro- mium 1 ; however, the long-term effects associated with exposure to metal ions have not been investi- gated. It has been reported that 97% of the chro- mium released on corrosion of stainless steel implants is in its hexavalent form. 2 This ion has been shown to cause direct toxicity to the cells of the immune system. It induces oxidative damage and apoptosis in lymphocytes and in macrophages. 3–5 Although the mechanisms involved in osteolysis induced by metal ions/particles are unclear, there are reports suggesting that tissue damage by necro- sis 6 and cell death by apoptosis 7 occur at the site of the implant. In addition to these overtly toxic effects, the release of metal ions from MOM can also induce an adaptive response in cells, a process that may be influenced by or may influence inflammatory mechanisms. Once released from an implant, Cr (VI) can enter the circulation. It can cross cell membranes via non- specific anionic channels and is reduced to Cr (III) by intracellular reductants (enzymatic and nonenzy- matic), in the process generating reactive intermedi- ates and oxygen species (ROS). The reduction of Cr (VI) by reduced glutathione (GSH) and glutathione reductase has been well documented. 8 These reactive intermediates have been postulated to interfere with cellular signaling cascades, cell proliferation, and activation of cytokines and transcription factors. 9 Cr (VI) is known to lead to toxicity in various cell types in vitro after acute exposure. 4,10 Various bio- chemical and molecular parameters of toxicity have been measured previously. Chromate compounds have been shown to cause cell-cycle arrest, inhibit expression of heme oxygenase-1, 11 disrupt the actin cytoskeleton 12 and cause apoptosis via ROS medi- Correspondence to: M. H. Grant; e-mail: m.h.grant@strath. ac.uk Contract grant sponsor: DePuy International Ltd. Ó 2009 Wiley Periodicals, Inc.