Jens Mattow 1, 2 * Ilja Demuth 1 * Gisela Haeselbarth 3 Peter R. Jungblut 2 Joachim Klose 1 1 Institute of Human Genetics, Charité, Berlin, Germany 2 Max Planck Institute for Infection Biology, Berlin, Germany 3 Institute for Biochemistry, Technical University, Berlin, Germany Received September 19, 2005 Revised November 3, 2005 Accepted November 3, 2005 Research Article Selenium-binding protein 2, the major hepatic target for acetaminophen, shows sex differences in protein abundance Liver samples from female and male mice of two subspecies, Mus musculus musculus and Mus musculus domesticus, were investigated by a combination of 2-DE and MALDI-MS. The image analysis of the generated 2-DE patterns revealed several pro- tein spots with significant differences in intensity/abundance between the sexes. Seven protein spots, which were prominent in 2-DE patterns of male mice, but which showed very low intensities in females, were identified as selenium-binding protein 2 (SBP2) also known as 56-kDa acetaminophen-binding protein. Edman degradation indicated that at least three of these protein spots represent N-terminally truncated SBP2 variants. Furthermore, it was shown that the observed differences in SBP2 abundance correlate with sex differences in transcription of the gene encoding SBP2, selenbp2, as revealed by RT-PCR and restriction digest as well as sequence analysis of the products. Since SBP2 has been described as the major target for acetaminophen in mouse liver cytosol, these findings are discussed with respect to their possible relevance for sex differences in acetaminophen-mediated toxicity, which have been described in a variety of mammals including mice and rats. Keywords: Liver / Mouse / Proteomics / Selenium-binding protein 2 / Sex differences DOI 10.1002/elps.200500703 1 Introduction Acetaminophen (paracetamol, N-acetyl-p-aminophenol, APAP) is a widely used analgesic and antipyretic. An acute overdose of the drug can cause severe liver and kidney damage and even death [1]. Comprehensive reviews on APAP metabolism and toxicity in mice and humans have recently been published [2–4]. The toxicity of APAP is closely linked to its metabolism. After ingestion of therapeutic doses, most APAP is modified in the liver by glucuronidation and sulfation to nontoxic agents, which are then eliminated in the urine. Under such condi- tions, only ,5% of the drug is metabolized by hepatic cytochrome P450 enzymes, including CYP1A2, CYP2E1, and CYP3A4, to the highly reactive metabolite N-acetyl- p-benzoquinone imine (NAPQI). After therapeutic APAP doses, NAPQI is efficiently inactivated, principally by reaction with reduced glutathione (GSH), and excreted. However, when APAP is taken/given in excess, the sulfa- tion and glucuronidation pathways become saturated, resulting in increased NAPQI formation. Under these conditions, GSH is depleted. NAPQI then accumulates and covalently binds to target proteins, forming APAP- protein adducts, as well as to the lipid bilayer of hepato- cytes, leading finally to centrilobular necrosis and sub- sequent liver and kidney dysfunction. It has been demonstrated in mice and humans that covalent binding of NAPQI to hepatic proteins (arylation) is highly selective, and that there is a strong relationship between arylation and APAP toxicity [3, 5–8]. Numerous hepatic target pro- teins of APAP have been identified, mainly by combina- Correspondence: Dr. Jens Mattow, Department of Immunology, Max Planck Institute for Infection Biology, Schumannstr. 21/22, 10117 Berlin, Germany E-mail: mattow@mpiib-berlin.mpg.de Fax: 149-30-28460-503 Abbreviations: acc., accession; APAP , acetaminophen; NAPQI, N- acetyl-p-benzoquinone imine; SBP , selenium-binding protein; selenbp, gene encoding selenium-binding protein Electrophoresis 2006, 27, 1683–1691 1683 * These authors contributed equally to this work.  2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.electrophoresis-journal.com