Prolonged treatment with N-acetylcystine delays liver recovery from acetaminophen hepatotoxicity

Abstract

Introduction: Acetaminophen (APAP) toxicity is the most common cause of acute liver failure in the US and Europe. Massive hepatocyte necrosis is the predominant feature of APAP-induced acute liver injury (ALI). Liver regeneration is a vital process for survival after a toxic insult, it occurs at a relative late time point after the injurious phase. Currently, N-acetylcysteine (NAC), a glutathione precursor, is the antidote for acetaminophen overdose. However, NAC is effective only for patients who present within hours of an acute overdose, and is less effective for late-presenting patients. It is possible that in delayed patients, previously reduced endogenous glutathione (GSH) level has restored and prolonged treatment with NAC might be toxic and impair liver regeneration. Therefore, we hypothesize that prolonged treatment with NAC impairs liver regeneration in ALI induced by APAP.

Methods: ALI was induced in C57BL/6 male mice by a single dose of APAP (350 mg/kg) by intraperitoneal injection. After two hours of APAP challenge, the mice were given 100 mg/kg NAC dissolved in 0.6 mL saline, or saline treatment every 12 hours for a total of 72 hours.

Results: Seventy-two hours after APAP challenge, compared with saline treatment, NAC treatment significantly increased serum transaminases (alanine transaminase/aspartate aminotransferase), induced evident hepatocyte vacuolation in the periportal area and delayed liver regeneration seen in histopathology. This detrimental effect was associated with reduced hepatic nuclear factor (NF)-kappaB DNA binding and decreased expression of cell cycle protein cyclin D1, two important factors in liver regeneration.

Conclusions: Prolonged treatment with NAC impairs liver regeneration in ALI induced by APAP.

Figure 1

Effect of treatment with NAC or saline on serum ALT/AST in an APAP-induced ALI model. (a, b) Acute liver injury (ALI) was induced in C57Bl/6 male mice with a single dose of acetaminophen (APAP) (350 mg/kg) by intraperitoneal injection. Two hours after APAP injection, the animals were treated with 100 mg/kg N-acetyl-cysteine (NAC) dissolved in 0.6 mL saline or 0.6 mL saline every 12 hours. Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were measured 24 hours after APAP injection (n = 6 surviving mice for each group). Results are means ± standard error of the mean (SEM). * P < 0.05 versus control; † P < 0.05 vs. the NAC group. (c, d) Three separate groups of mice were used. ALI was induced as described above. Two hours after APAP challenge, the animals were given the same treatment every 12 hours for a total of 72 hours. ALT and AST were measured 72 hours after APAP injection (n = 6 to 7 surviving mice for each group). Results are means ± SEM. * P < 0.05 vs. control; † P < 0.05 vs. the NAC group.

Figure 2

Effect of treatment with NAC or saline on pathology in mice with ALI. H&E staining was assessed 72 hour after induction of acute liver injury (ALI) or sham procedure (n = 6 for each group). Typical picture is shown. Arrows indicate periportal areas shown in Figure 3. NAC = N-acetyl-cysteine.

Figure 3

Prolonged treatment with NAC induces periportal hepatocytes vacuolation in mice with ALI. H&E staining was assessed 72 hour after induction of acute liver injury (ALI) or sham procedure (n = 6 for each group). Typical picture is shown. NAC = N-acetyl-cysteine.

Figure 4

Effect of treatment with NAC or saline on hepatic MPO activity in mice with ALI. Liver myeloperoxidase (MPO) was assessed 72 hours after induction of acute liver injury (ALI) or sham procedure. Results are means ± standard error of the mean (SEM). * P < 0.05 versus control; † P < 0.05 vs. saline. NAC = N-acetyl-cysteine.

Figure 5

Effect of treatment with NAC or saline on NF-κB DNA binding in nuclear extracts prepared from hepatic tissue samples from mice with ALI. Nuclear factor (NF) κB DNA binding was assessed 72 hours after induction of acute liver injury (ALI) or sham procedure. The figure depicts results from five representative assays. Typical gels are depicted. NAC = N-acetyl-cysteine.

Figure 6

Effect of treatment with NAC or saline on the expression of cyclin D1 in the hepatic tissue. Western blot was performed using hepatic extracts prepared from tissues obtained 72 hours after acetaminophen injection. The figure depicts results from five representative assays. Typical gels are depicted. NAC = N-acetyl-cysteine.