Toxicology Letters 221 (2013) 219–224 Contents lists available at SciVerse ScienceDirect Toxicology Letters jou rn al hom epage: www.elsevier.com/locate/toxlet Biomarkers for methotrexate-induced liver injury: Urinary protein profiling of psoriasis patients Rachel P.L. van Swelm a , Coby M.M. Laarakkers b , Marisol Kooijmans-Otero c , Elke M.G.J. de Jong c , Rosalinde Masereeuw a , Frans G.M. Russel a,∗ a Department of Pharmacology and Toxicology, Radboud University Nijmegen Medical Centre, P.O. Box 9101, 6500 HB Nijmegen, The Netherlands b Department of Laboratory Medicine, Radboud University Nijmegen Medical Centre, P.O. Box 9101, 6500 HB Nijmegen, The Netherlands c Department of Dermatology, Radboud University Nijmegen Medical Centre, P.O. Box 9101, 6500 HB Nijmegen, The Netherlands h i g h l i g h t s • High cumulative MTX dose in psoriasis patients is associated with hepatic fibrosis. • Urine proteomics shows a differentiating profile after high cumulative MTX dose. • Urine of psoriasis patients using MTX contains proteins related to hepatic fibrosis. • Urinary biomarkers related to fibrosis might be used to monitor MTX hepatotoxicity. a r t i c l e i n f o Article history: Received 30 April 2013 Received in revised form 21 June 2013 Accepted 24 June 2013 Available online 3 July 2013 Keywords: Methotrexate MALDI-TOF MS Biomarker Hepatic fibrosis N-cadherin a b s t r a c t Hepatic fibrosis is an adverse drug reaction of methotrexate (MTX) seen after long-term use in psoriasis patients. Currently, patients are monitored for MTX-induced hepatic fibrosis by performing liver biopsy, which is risky and burdensome for the patient, or by measuring plasma procollagen type III aminopeptide (PIIINP), which is not conclusive. The objective of this study was to identify novel predictive and preferably non-invasive biomarkers to monitor psoriasis patients for MTX-induced hepatic fibrosis. Urine samples were collected from 60 psoriasis patients treated with MTX and divided into two categories: low cumulative dose (<1500 mg MTX) and high cumulative dose (>1500 mg). Urinary pro- teins were profiled using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and identified using electrospray ionization LTQ. In urine of psoriasis patients with high cumulative MTX dose multiple proteins were identified that are associated with hepatic fibrosis, such as N-cadherin, inter-alpha-trypsin inhibitor heavy chain H4, haptoglobin and serotransferrin. These proteins may be candidate urinary biomarkers to monitor MTX-induced hepatic fibrosis. In conclusion, urinary proteome analysis identified a profile of potentially predictive biomarkers for MTX-induced hepatic fibrosis in psoriasis patients with high cumulative dose of MTX. © 2013 Elsevier Ireland Ltd. All rights reserved. 1. Introduction Methotrexate (MTX) is a folic acid antagonist with anti- proliferative and anti-inflammatory properties that has been used for the treatment of rheumatoid arthritis (RA) and psoriasis (Shen Abbreviations: ALT, alanine aminotransferase; ESI, electrospray ionization; ITIH4, inter-alpha-trypsin inhibitor heavy chain H4; MALDI-TOF MS, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry; MTX, methotrexate; PASI, psoriasis area and severity index; PIIINP, procollagen type III aminopeptide; RA, rheumathoid arthritis. ∗ Corresponding author at: Department of Pharmacology and Toxicology (149), Radboud University Nijmegen Medical Centre, P.O. Box 9101, 6500 HB Nijmegen, The Netherlands. Tel.: +31 243616892; fax: +31 243614214. E-mail address: F.Russel@pharmtox.umcn.nl (F.G.M. Russel). et al., 2012). Despite adverse reactions, including liver injury, MTX has been prescribed for decades because of its effectiveness (Warren et al., 2008). MTX-induced liver injury is characterized by hepatic fibrosis, which is reported more often in psoriasis patients than RA patients (Taylor et al., 2008). Long-term low dose MTX treatment results in progression of liver disease in 33% of psoriasis patients (Aithal, 2007; Bray et al., 2012; Yeo et al., 2013), although a relation with non-alcoholic steatosis hepatitis (NASH), which in turn is related to diabetes mellitus and high body mass index (BMI), seems to be also present (Miele et al., 2009; Rosenberg et al., 2007). The risk of MTX-induced hepatic fibrosis increases with treatment duration and, typically, most cases of hepatic fibrosis present at a cumulative MTX dose between 1500 and 6000 mg (Berends et al., 2006). Risk factors for MTX-induced fibrosis are obesity and dia- betes mellitus type 2, and to a lesser extent alcohol consumption 0378-4274/$ – see front matter © 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.toxlet.2013.06.234