S collected in lithium-heparin tubes by eye extraction under Indolactam V 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 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 1480666 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 were determined at hospital admission (plasma sample 1) and within 24 h after admission (plasma sample 2). Plasma 1676428 liver enzyme values of both plasma samples were substantially increased. Enzyme concentrations in sample 1 and sample 2 were: ALT 8475 U/L and 9265 U/L (reference value ,35), aspartate aminotransferase 16850 U/L and 18420 U/L (ref ,40), lactate dehydrogenase 16010 u/L andUrinary Biomarkers of Acetaminophen HepatotoxicityUrinary Biomarkers of Acetaminophen HepatotoxicityFigure 1. APAP-induced liver injury and kidney histology in mice. Hematoxylin and eosin staining of representative liver slides from a vehicle-treated mouse (A and C) and an APAP-treated mouse (B and D). Panels A and B show a 106 magnification, and a 206 magnification of the framed area is given in panels C and D, respectively. Centrilobular necrosis can be observed in liver slides after APAP treatment. Plasma ALT levels (E) and the percentage of centrilobular necrosis (F) increased significantly in mice receiving 275 and 350 mg/kg APAP. Thiazole Orange site Periodic acid-Schiff staining of representative kidney slides from a vehicle-treated mouse (G and I) and an APAP-treated mouse (H and J) show no difference in histology. Panels G and H demonstrate a 206 magnification and a 406 magnification is given for the framed areas in panels I and J. The scalebar represents 200 mm in the slides with 106 magnification, 100 mm with 206 magnification and 50 mm with 406 magnification. ** P,0.01, *** P,0.001 compared to vehicle treated mice. ALT: alanine aminotransferase; AMAP: 3-acetamidophenol; APAP: acetaminophen. doi:10.1371/journal.pone.0049524.g17730 U/L (.S 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 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 1480666 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 were determined at hospital admission (plasma sample 1) and within 24 h after admission (plasma sample 2). Plasma 1676428 liver enzyme values of both plasma samples were substantially increased. Enzyme concentrations in sample 1 and sample 2 were: ALT 8475 U/L and 9265 U/L (reference value ,35), aspartate aminotransferase 16850 U/L and 18420 U/L (ref ,40), lactate dehydrogenase 16010 u/L andUrinary Biomarkers of Acetaminophen HepatotoxicityUrinary Biomarkers of Acetaminophen HepatotoxicityFigure 1. APAP-induced liver injury and kidney histology in mice. Hematoxylin and eosin staining of representative liver slides from a vehicle-treated mouse (A and C) and an APAP-treated mouse (B and D). Panels A and B show a 106 magnification, and a 206 magnification of the framed area is given in panels C and D, respectively. Centrilobular necrosis can be observed in liver slides after APAP treatment. Plasma ALT levels (E) and the percentage of centrilobular necrosis (F) increased significantly in mice receiving 275 and 350 mg/kg APAP. Periodic acid-Schiff staining of representative kidney slides from a vehicle-treated mouse (G and I) and an APAP-treated mouse (H and J) show no difference in histology. Panels G and H demonstrate a 206 magnification and a 406 magnification is given for the framed areas in panels I and J. The scalebar represents 200 mm in the slides with 106 magnification, 100 mm with 206 magnification and 50 mm with 406 magnification. ** P,0.01, *** P,0.001 compared to vehicle treated mice. ALT: alanine aminotransferase; AMAP: 3-acetamidophenol; APAP: acetaminophen. doi:10.1371/journal.pone.0049524.g17730 U/L (.
