Toxicology Letters 224 (2014) 54–63 Contents lists available at ScienceDirect Toxicology Letters jou rn al hom ep age: www.elsevier.com/locate/toxlet Alternative biomarkers of n-hexane exposure: Characterization of aminoderived pyrroles and thiol-pyrrole conjugates in urine of rats exposed to 2,5-hexanedione M. Edite Torres a,b , Luísa L. Gonc ¸ alves b , M. Rosário Bronze a , A.P. Marreilha dos Santos a , M. Camila Batoréu a , M. Luísa Mateus a,∗ a iMed.UL, Department of Toxicology and Food Sciences, Faculty of Pharmacy, University of Lisbon, Av. Prof. Gama Pinto, 1649-003 Lisbon, Portugal b CiiEM, Center for Interdisciplinary Research Egas Moniz, ISCSEM, Institute of Health and Life Sciences Egas Moniz, Campus Universitário, Quinta da Granja, 2829-511 Caparica, Portugal a r t i c l e i n f o Article history: Received 4 August 2013 Received in revised form 8 October 2013 Accepted 11 October 2013 Available online xxx Keywords: 2,5-Hexanedione Pyrrole derivatives Urinary biomarkers LC–MS/MS a b s t r a c t The identification of pyrrole derivatives in urine of rats exposed to 2,5-hexanedione (2,5-HD), was performed to select an adequate peripheral biomarker predictive of 2,5-HD neurotoxicity. Studies on molecular mechanism of 2,5-HD neurotoxicity have revealed that 2,5-hexanedione reacts with free amino groups of lysine in proteins forming primary pyrrole adducts, which may autoxidize and form pyrrole dimers, responsible for protein crosslinking in neurofilaments, or react with sulfhydryl groups of cysteine in peptides and proteins, forming secondary pyrrole adducts, which probably may inhibit the process responsible by 2,5-HD neurotoxicity. In this work, the analysis of excreted 2,5-HD and pyr- role derivatives in urine of rats i.p. treated with 3 doses of 2,5-HD (400 mg/kg bw/48 h) was performed using ESI-LC–MS/MS. Several pyrrole compounds were identified, namely dimethylpyrrole norleucine (DMPN), cysteine-pyrrole conjugate (DMPN NAC), glutathione-pyrrole conjugate (DMPN GSH) and 2,5- dimethylpyrrole (2,5-DMP). Additionally, free and total 2,5-HD, DMPN and DMPN NAC were quantified. The observed results suggest that DMPN is a sensitive and specific indicator of repeated exposure to 2,5-HD. © 2013 Elsevier Ireland Ltd. All rights reserved. 1. Introduction Chronic exposure to n-hexane causes a neurotoxic condition classified as axonal atrophy in peripheral nervous system (PNS) and central nervous system (CNS) in humans and experimental animals (Yamada, 1964; Spencer et al., 1978; Lehning et al., 2000; LoPachin and DeCaprio, 2004; Zhang et al., 2010). In vivo and in vitro models demonstrated that the ulti- mate neurotoxic metabolite of this solvent is 2,5-hexanedione (2,5-HD) (DeCaprio et al., 1983; Zhu et al., 1993) which metabolic pathway of detoxification leads to the formation of Abbreviations: 2,5-DMP, 2,5-dimethylpyrrole; 2,5-HD, 2,5-hexanedione; BEI, Biological Exposure Indices; DMPN GSH, glutathione-pyrrole conjugate; DMPN NAC, cysteine-pyrrole conjugate; DMPN, dimethylpyrrole norleucine; ESI-LC–MS/MS electrospray ionization, liquid chromatography–mass spectroscopy; GSH, -l- glutamyl-l-cysteinyl-glycine; m/z, mass-to-charge ratio; MRM, multiple reactions monitoring; NAC, N-acetyl-l-cysteine; NAL, N˛-acetyl-l-lysine; p-DMAB, 4- dimethylaminobenzaldheyde; PLS, Pyrrole Like Substances; SRM, Selected Reaction Monitoring; TWA, time-weighted average. ∗ Corresponding author. Tel.: +351 217946400; fax: +351 217946470. E-mail address: lmateus@ff.ul.pt (M.L. Mateus). 4,5-dihydroxy-2-hexanone, excreted in urine as a glucuronide conjugated form (Fedtke and Bolt, 1987). Therefore, urinary levels of total 2,5-HD (free 2,5-HD + 4,5-dihydroxi-2-hexanone) have been used in routine analysis as a biomarker of occupational exposure to n-hexane (Perbellini et al., 1990; Biological Exposure Values for Occupational Toxicants and Carcinogens, 1994). In 2001 ACGIH changed the Biological Exposure Index (BEI) for 2,5-HD, rec- ommending the determination of “free” instead of “total” 2,5-HD with a BEI value of 0.4 mg/g creatinine, corresponding to a Thresh- old Weight Average, (TWA), of 50 ppm (American Conference of Governmental Industrial Hygienists, 2004). In fact, “free” 2,5-HD is a good analytical and biological biomarker of n-hexane exposure, that may indicate the amount of 2,5-HD that escapes to the detox- ification process, thus, being a better predictor of the neurotoxic risk than the conjugated metabolites that are rapidly excreted in urine (Manini et al., 1999, 2004; Mateus et al., 2001). Mechanistic studies showed that 2,5-HD, a  -diketone, when administered to experimental animals, can form adducts with numerous proteins, in a selective manner, binding to amino groups of lysine in axonal cytoskeletal proteins and forming 2,5-DMP (2,5-dimethylpyrrole) primary adducts, within specific regions of neurofilaments (NFs) (DeCaprio and Fowke, 1992; DeCaprio 0378-4274/$ – see front matter © 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.toxlet.2013.10.011