Esented as mean 6 SD. doi:10.1371/journal.pone.0049524.tproteins involved in lipid/fatty acid metabolism, energy metabolism, oxidative stress, calcium homeostasis and inflammation. The goal of this study was to identify proteins in human urine related to acute DILI. To this end, we implemented a translational approach to identify urinary biomarkers for human DILI. By first identifying proteins related to liver injury in urine of mice exposed to the drug of interest, and subsequently searching for the orthologous proteins in human urine, we aim to more efficiently use the limited availability of human urine samples for biomarker assessment. Here, we show carbonic anhydrase 3 (CA3), superoxide dismutase 1 (SOD1) and calmodulin (CaM) as potential urinary biomarkers for APAP-induced liver injury in both mouse and human.Animal experimentMale FVB mice (Charles River, Germany; 22?8 g bw) were housed under controlled conditions and randomly assigned to a single i.p. injection of vehicle (saline, n = 19)) or 100 (n = 6), 225 (n = 18), 275 (n = 33) or 350 (n = 6) mg/kg bw APAP (A500 SigmaAldrich Chemie B.V., Zwijndrecht, the Netherlands). As a negative control, mice (n = 6) were treated with 350 mg/kg bw 3-acetamidophenol (AMAP; A7205, Sigma-Aldrich). After injection, mice were placed individually in metabolic cages (Techniplast, Germany GmbH) to collect 24 h urine samples, with water and pulverized standard chow ad libitum. Protease inhibitors (Complete Mini, Roche Diagnostics, Almere, the Netherlands) were added to the urine, which was then centrifuged at 30006 g for 10 min at 4uC. Subsequently, blood plasma was collected in lithium-heparin tubes by eye extraction under isoflurane anesthesia and animals were sacrificed by cervical dislocation. Urine creatinine and plasma ALT levels were assessed by routine assays.Materials and Methods Ethics statementAll experiments were approved by the local Animal Welfare Committee 15755315 of the Radboud University Nijmegen (RU-DEC 2008142 and RU-DEC 2009-101), in accordance with the guidelines of the Principles of Laboratory Animal Care (NIH publication 86-23, revised 1985). Human sample collection was evaluated by the ethical committee of the Radboud University Nijmegen Medical Centre and the Hagaziekenhuis (Den Haag, the Netherlands) and they concluded that the performed research was not conducted under the regulations of the Act on Medical Research Involving Human Subjects, because sample collection included non-invasive sampling of urine and use of leftover plasma samples, taken for clinical analysis. Moreover, samples were collected anonymously and no clinically relevant or incriminating information were used. Written informed consent, therefore, was not compulsory; however, oral informed consent was obtained for all volunteers, patients and the parents of the underage patient with SR3029 site acetaminophen intoxication, which was not recorded to keep the 
procedure anonymous.Human sample collectionFirst, a Calciferol control master pool was created consisting of 24 urine samples of both male and female volunteers between 18?5 years of age. Next, we were able to collect urine of a severe APAP intoxication, concerning a 5 year old girl of 12.5 kg bw that ingested approximately 12 tablets of 500 mg APAP. We received one urine sample collected upon hospital admission (urine sample 1) and one pooled urine sample composed of urine collected previous to, during, and after N-acetyl cysteine treatment (urine sample 2). Plasma liver enzymes we.Esented as mean 6 SD. doi:10.1371/journal.pone.0049524.tproteins involved in lipid/fatty acid metabolism, energy metabolism, oxidative stress, calcium homeostasis and inflammation. The goal of this study was to identify proteins in human urine related to acute DILI. To this end, we implemented a translational approach to identify urinary biomarkers for human DILI. By first identifying proteins related to liver injury in urine of mice exposed to the drug of interest, and subsequently searching for the orthologous proteins in human urine, we aim to more efficiently use the limited availability of human urine samples for biomarker assessment. Here, we show carbonic anhydrase 3 (CA3), superoxide dismutase 1 (SOD1) and calmodulin (CaM) as potential urinary biomarkers for APAP-induced liver injury in both mouse and human.Animal experimentMale FVB mice (Charles River, Germany; 22?8 g bw) were housed under controlled conditions and randomly assigned to a single i.p. injection of vehicle (saline, n = 19)) or 100 (n = 6), 225 (n = 18), 275 (n = 33) or 350 (n = 6) mg/kg bw APAP (A500 SigmaAldrich Chemie B.V., Zwijndrecht, the Netherlands). As a negative control, mice (n = 6) were treated with 350 mg/kg bw 3-acetamidophenol (AMAP; A7205, Sigma-Aldrich). After injection, mice were placed individually in metabolic cages (Techniplast, Germany GmbH) to collect 24 h urine samples, with water and pulverized standard chow ad libitum. Protease inhibitors (Complete Mini, Roche Diagnostics, Almere, the Netherlands) were added to the urine, which was then centrifuged at 30006 g for 10 min at 4uC. Subsequently, blood plasma was collected in lithium-heparin tubes by eye extraction under isoflurane anesthesia and animals were sacrificed by cervical dislocation. Urine creatinine and plasma ALT levels were assessed by routine assays.Materials and Methods Ethics statementAll experiments 
were approved by the local Animal Welfare Committee 15755315 of the Radboud University Nijmegen (RU-DEC 2008142 and RU-DEC 2009-101), in accordance with the guidelines of the Principles of Laboratory Animal Care (NIH publication 86-23, revised 1985). Human sample collection was evaluated by the ethical committee of the Radboud University Nijmegen Medical Centre and the Hagaziekenhuis (Den Haag, the Netherlands) and they concluded that the performed research was not conducted under the regulations of the Act on Medical Research Involving Human Subjects, because sample collection included non-invasive sampling of urine and use of leftover plasma samples, taken for clinical analysis. Moreover, samples were collected anonymously and no clinically relevant or incriminating information were used. Written informed consent, therefore, was not compulsory; however, oral informed consent was obtained for all volunteers, patients and the parents of the underage patient with acetaminophen intoxication, which was not recorded to keep the procedure anonymous.Human sample collectionFirst, a control master pool was created consisting of 24 urine samples of both male and female volunteers between 18?5 years of age. Next, we were able to collect urine of a severe APAP intoxication, concerning a 5 year old girl of 12.5 kg bw that ingested approximately 12 tablets of 500 mg APAP. We received one urine sample collected upon hospital admission (urine sample 1) and one pooled urine sample composed of urine collected previous to, during, and after N-acetyl cysteine treatment (urine sample 2). Plasma liver enzymes we.
