Plasma and liver acetaminophen-protein adduct levels in mice after acetaminophen treatment: dose-response, mechanisms, and clinical implications

Abstract

At therapeutic doses, acetaminophen (APAP) is a safe and effective analgesic. However, overdose of APAP is the principal cause of acute liver failure in the West. Binding of the reactive metabolite of APAP (NAPQI) to proteins is thought to be the initiating event in the mechanism of hepatotoxicity. Early work suggested that APAP-protein binding could not occur without glutathione (GSH) depletion, and likely only at toxic doses. Moreover, it was found that protein-derived APAP-cysteine could only be detected in serum after the onset of liver injury. On this basis, it was recently proposed that serum APAP-cysteine could be used as diagnostic marker of APAP overdose. However, comprehensive dose-response and time course studies have not yet been done. Furthermore, the effects of co-morbidities on this parameter have not been investigated. We treated groups of mice with APAP at multiple doses and measured liver GSH and both liver and plasma APAP-protein adducts at various timepoints. Our results show that protein binding can occur without much loss of GSH. Importantly, the data confirm earlier work that showed that protein-derived APAP-cysteine can appear in plasma without liver injury. Experiments performed in vitro suggest that this may involve multiple mechanisms, including secretion of adducted proteins and diffusion of NAPQI directly into plasma. Induction of liver necrosis through ischemia-reperfusion significantly increased the plasma concentration of protein-derived APAP-cysteine after a subtoxic dose of APAP. While our data generally support the measurement of serum APAP-protein adducts in the clinic, caution is suggested in the interpretation of this parameter.

Figure 1

Dose-response of liver injury. Mice were treated with 0, 15, 75, 150, 300, or 600 mg APAP / kg bodyweight and sacrificed 24 h later. (A) Dose-response of plasma alanine aminotransferase (ALT) activities. (B) Dose-response of plasma glutamate dehydrogenase (GDH) activities. (C) Hematoxylin and eosin staining of liver sections. (D) TUNEL staining of liver sections. Data are expressed as mean ± SEM of n = 6 animals per time point. *P < 0.05 (compared to t=0); NA = Not Available.

Figure 2

Dose-response and time course of total liver GSH (GSH + GSSG) and liver APAP-protein adducts. Mice were treated with 0, 15, 75, 150, 300, or 600 mg APAP / kg bodyweight and sacrificed at the indicated time points. (A) Total liver GSH over time. (B) Liver protein-derived APAP-CYS over time. (C) Total liver GSH 0.5 h after APAP treatment. (D) Liver proteinderived APAP-CYS concentration, 1 h after APAP treatment. Data are expressed as mean ± SEM of n = 6 animals per time point.

Figure 3

Dose-response and time course of plasma and kidney APAP-protein adducts. Mice were treated with 0, 15, 75, 150, 300, or 600 mg APAP / kg bodyweight and sacrificed at the indicated time points. Protein-derived APAP-CYS was measured in plasma (A and B) and kidneys (C and D) from these animals. Data are expressed as mean ± SEM of n = 6 animals per time point.

Figure 4

Time course of plasma APAP-protein adducts and liver injury after 75 mg/kg or 150 mg/kg dose. Mice were treated with 75 or 150 mg APAP / kg bodyweight and sacrificed at the indicated time points. (A,C) ALT and protein-derived APAP-CYS in plasma. (B,D) Liver histology time course. Data are expressed as mean ± SEM of n = 6 animals per time point.

Figure 5

Time course of plasma APAP-protein adducts and liver injury after 300 mg/kg or 600 mg/kg dose. Mice were treated with 300 or 600 mg APAP / kg bodyweight and sacrificed at the indicated time points. (A,C) ALT and protein-derived APAP-CYS in plasma. (B,D) Liver histology time course. Data are expressed as mean ± SEM of n = 6 animals per time point.

Figure 6

Mechanisms of the appearance of extracellular APAP-protein adducts. (A) Primary mouse hepatocytes were thoroughly washed and cultured in serum-free medium for 3 h after exposure to 5 mM APAP. Extracellular protein was measured using SDS-PAGE with Coomassie blue staining. (B) LDH release was measured in cultures of primary mouse hepatocytes treated with APAP in the presence or absence of fetal bovine serum (FBS) for 3 h. (C) Protein-derived APAP-CYS was measured in the culture medium of hepatocytes treated with APAP in the presence or absence of serum for 3 h. (D) Protein-derived APAP-CYS was measured in hepatocytes treated with APAP in the presence of absence of serum for 3 h. Data are expressed as mean ± SEM of 3–6 experiments. *P < 0.05 (compared to cultures with serum-free medium). ND = Not Detectable.

Figure 7

Ischemia-reperfusion liver injury increases plasma APAP-protein adduct levels. Mice were pretreated for 1 h with APAP at 75 mg/kg and subjected to ischemia-reperfusion of the liver. (A) Plasma ALT values. (B) Protein-derived APAP-CYS in plasma. (C) Protein-derived APAP-CYS in livers. (D) Total GSH (GSH + GSSG) in livers. Data are expressed as mean ± SEM of n = 4–6 animals per treatment group. *P < 0.05 (compared to untreated controls). ^#P < 0.05 (compared to sham). ND = Not Detectable.