Nanoparticulate silver increases uric acid and allantoin excretion in rats, as identified by metabolomics Niels Hadrup, a * Henrik R. Lam, a,b Katrin Loeschner, c Alicja Mortensen, a Erik H. Larsen c and Henrik Frandsen c ABSTRACT: Metabolomic investigation of rat urine was employed to identify mammalian metabolites affected by ionic or nanoparticulate silver. Female and male Wistar rats were administered silver nanoparticles (2.25, 4.5 or 9.0 mg kg 1 body weight per day) or ionic silver (silver acetate, 9.0 mg silver kg 1 bw per day) by oral gavage for 28 days. On day 18, urine was collected for 24 h and subjected to metabolomics with high performance liquid chromatography–quadropole time-of- flight mass spectrometry (HPLC-QTOF-MS)-based separation and detection. Principal component analysis was subsequently applied to the data. Metabolomic differences in urine composition were found in female rats but not in male rats. Several metabolites were identified by the use of elemental composition calculated from the exact mass combined with searches in the Human Metabolome Database.The metabolite identities were eventually verified by co-chromatography with authentic standards. Differences were found in uric acid and its degradation product, allantoin. Administration of nanoparticulate silver increased both metabolites, whereas ionic silver only increased allantoin. In conclusion, metabolomic investigation of rat urine showed that increased levels of uric acid and allantoin were associated with exposure to nanoparticulate silver. Copyright © 2012 John Wiley & Sons, Ltd. Keywords: toxicology; nanoparticle; silver; metabolomics; allantoin; uric acid INTRODUCTION For decades, ionic silver has been employed for antibacterial applications (Alexander, 2009). Silver nanoparticles (AgNPs) have been developed for the purpose of slow release of silver ions, thereby providing an extended anti-bacterial effect (Wijnhoven et al., 2009). Silver is used in consumer products either in its ionic form (e.g. wound dressings) or as AgNPs (e.g. in refrigerators, toothpastes and health drinks) (Woodrow Wilson International Center for Scholars, 2011). Therefore, it is important to investigate the potential physiological, pharmacological and toxicological properties of silver. Conventional toxicological studies have shown that toxic doses of ionic silver cause such effects as loss of body weight, liver damage, argyria (irreversible blue-gray discoloration of the skin) and, ultimately, death (Buckley and Terhaar, 1973; Walker, 1971; Hanna et al., 1974; Rich et al., 1972). Silver in the form of AgNPs has displayed only minimal toxicity in animal studies (Kim et al., 2008, 2010; Hadrup et al., 2012). Overall, limited knowledge is available on the mechanism of action of both ionic and nanoparticulate silver in mammals. ‘Omics’ techniques can be used to screen for new unexpected effects and toxicological mechanisms of chemicals. Metabolomics is the concomitant measurement of a wide range of low- molecular-weight molecules in body fluids such as plasma or urine. This technique has previously been employed in the investigation of non-AgNP effects. Bu et al. (2010) used NMR-based metabolomics to investigate the effects of TiO 2 -NPs administered orally to rats. Urine and serum were investigated, and several changes in metabolite concentrations were detected. Feng and coworkers investigated the effects of intravenous iron NP administration on the metabolome in rat plasma and urine. NMR- based analysis showed a wide range of changes in metabolite levels (Feng et al., 2010). Lei et al. (2008) investigated the effects of oral dosing of rats with copper NPs. NMR-based metabolomic analysis of urine, serum, liver and kidney extracts showed a change in the concentrations of a large number of metabolites.Using high- performance liquid chromatography–mass spectrometry (HPLC- MS)- based metabolomic analysis, Lu et al. (2011) investigated the effect of silica NPs on the biochemical composition of liver tissue and serum from mice injected intravenously. The authors found that the concentration of a range of metabolites were altered. These investigations, except for that of Lu et al., did not report the use of authentic standards to verify the identity of the metabolites. In this study, we employ metabolomic investigation with HPLC separation and quadropole time-of-flight (QTOF) accurate mass detection to investigate the whole metabolome in the urine of rats dosed orally for 18 days with 14 nm AgNPs at 2.25, 4.5 or 9 mg kg 1 body weight (bw) per day or with ionic silver *Correspondence to: Niels Hadrup, Division of Toxicology and Risk Assessment, National Food Institute, Technical University of Denmark, Room 116, Building G, Mørkhøj Bygade 19, DK-2860, Søborg, Denmark. E-mail: niels.hadrup@gmail.com a Division of Toxicology and Risk Assessment, National Food Institute, Technical University of Denmark, Mørkhøj Bygade 19, DK-2860, Søborg, Denmark b Environment and Toxicology, DHI, Agern Allé 5, DK-2970, Hørsholm, Denmark c Division of Food Chemistry, National Food Institute, Technical University of Denmark J. Appl. Toxicol. 2012 Copyright © 2012 John Wiley & Sons, Ltd. Research Article Received: 2 February 2012, Revised: 18 April 2012, Accepted: 18 April 2012 Published online in Wiley Online Library (wileyonlinelibrary.com) DOI 10.1002/jat.2779