Ers, because they vary in many features. For example, nutritional status, disease condition, and/or use of other drugs may affect the urinary proteome. Using a translational approach, 
we were able to identify potential bioMedChemExpress 69056-38-8 markers for APAP-induced liver SMER28 injury in mice and confirm the presence of these proteins in human urine samples after APAP intoxication and DILI caused by other drugs. In mice, urine was collected during 24 h after APAP administration, and plasma and liver tissue samples at 24 h after exposure. We measured urine at one time point after APAP administration, but still observed a strong association between plasma ALT values and both SOD1 and CaM levels in urine samples. Yet, we could 
not assess if these potential biomarkers are excreted in urine early after the onset of injury. Nevertheless, SOD1 has previously been reported to appear in rat urine as early as 12 h after treatment with CCl4, another known hepatotoxic chemical [22]. A disadvantage of urine collection during 24 h could be that potentially interesting proteins are difficult to detect because of dilution, particularly those excreted shortly after theUrinary Biomarkers of Acetaminophen Hepatotoxicityonset of injury. In addition, some proteins may be unstable in urine and only fragments rather than intact proteins can be detected. This has likely occurred for CA3 in the present study. Obviously, the kidney has a major influence on urine content and approximately 70 of the proteins in urine originate from this organ [23]. Since most proteins identified in this study are not liver-specific, we investigated whether potential kidney injury by APAP could have been a confounding factor. No signs of kidney injury were observed after APAP treatment as determined by histology and the absence of kidney injury markers (kidney injury molecule-1 and neutrophil gelatinase associated lipocalin; data not shown). We, therefore, assume that the proteins found in urine after APAP-induced liver injury were not the result of kidney injury, but were released from liver into blood and subsequently excreted by the kidney. Most of the proteins identified in this study were only found in mice with high plasma ALT values and do not seem to be suitable as biomarker. Urinary CA3 and SOD1 showed a good correlation with plasma ALT and probably are also leakage markers of injured hepatocytes. The advantage over plasma ALT is that these markers can be measured in patients non-invasively. CaM proved to be the most promising biomarker, because the protein was found in urine of mice treated with a high dose of APAP that did not show elevated plasma ALT levels. This was also observed in urine samples of human APAP intoxicants. Although plasma ALT levels were not increased in these patients, plasma APAP concentrations were high enough that liver injury was a concern as indicated by the Rumack-Matthew normogram 1326631 [24]. These data indicate that CaM has potential as predictive biomarker for acute DILI and that a mechanism of hepatocyte release other than leakage may be involved. Most of the proteins that we detected in urine are involved in intracellular processes related to APAP-induced liver injury (Table 1 and 2) [25,26,27,28]. These process are not specific to APAP and, accordingly, the biomarkers identified in this study are most likely not specific to APAP, but rather to acute hepatocellular injury. In line with this, urinary CaM concentration was also increased in human cases of DILI not caused b.Ers, because they vary in many features. For example, nutritional status, disease condition, and/or use of other drugs may affect the urinary proteome. Using a translational approach, we were able to identify potential biomarkers for APAP-induced liver injury in mice and confirm the presence of these proteins in human urine samples after APAP intoxication and DILI caused by other drugs. In mice, urine was collected during 24 h after APAP administration, and plasma and liver tissue samples at 24 h after exposure. We measured urine at one time point after APAP administration, but still observed a strong association between plasma ALT values and both SOD1 and CaM levels in urine samples. Yet, we could not assess if these potential biomarkers are excreted in urine early after the onset of injury. Nevertheless, SOD1 has previously been reported to appear in rat urine as early as 12 h after treatment with CCl4, another known hepatotoxic chemical [22]. A disadvantage of urine collection during 24 h could be that potentially interesting proteins are difficult to detect because of dilution, particularly those excreted shortly after theUrinary Biomarkers of Acetaminophen Hepatotoxicityonset of injury. In addition, some proteins may be unstable in urine and only fragments rather than intact proteins can be detected. This has likely occurred for CA3 in the present study. Obviously, the kidney has a major influence on urine content and approximately 70 of the proteins in urine originate from this organ [23]. Since most proteins identified in this study are not liver-specific, we investigated whether potential kidney injury by APAP could have been a confounding factor. No signs of kidney injury were observed after APAP treatment as determined by histology and the absence of kidney injury markers (kidney injury molecule-1 and neutrophil gelatinase associated lipocalin; data not shown). We, therefore, assume that the proteins found in urine after APAP-induced liver injury were not the result of kidney injury, but were released from liver into blood and subsequently excreted by the kidney. Most of the proteins identified in this study were only found in mice with high plasma ALT values and do not seem to be suitable as biomarker. Urinary CA3 and SOD1 showed a good correlation with plasma ALT and probably are also leakage markers of injured hepatocytes. The advantage over plasma ALT is that these markers can be measured in patients non-invasively. CaM proved to be the most promising biomarker, because the protein was found in urine of mice treated with a high dose of APAP that did not show elevated plasma ALT levels. This was also observed in urine samples of human APAP intoxicants. Although plasma ALT levels were not increased in these patients, plasma APAP concentrations were high enough that liver injury was a concern as indicated by the Rumack-Matthew normogram 1326631 [24]. These data indicate that CaM has potential as predictive biomarker for acute DILI and that a mechanism of hepatocyte release other than leakage may be involved. Most of the proteins that we detected in urine are involved in intracellular processes related to APAP-induced liver injury (Table 1 and 2) [25,26,27,28]. These process are not specific to APAP and, accordingly, the biomarkers identified in this study are most likely not specific to APAP, but rather to acute hepatocellular injury. In line with this, urinary CaM concentration was also increased in human cases of DILI not caused b.