Delayed administration of N-acetylcysteine blunts recovery after an acetaminophen overdose unlike 4-methylpyrazole

Abstract

N-acetylcysteine (NAC) is the only clinically approved antidote against acetaminophen (APAP) hepatotoxicity. Despite its efficacy in patients treated early after APAP overdose, NAC has been implicated in impairing liver recovery in mice. More recently, 4-methylpyrazole (4MP, Fomepizole) emerged as a potential antidote in the mouse APAP hepatotoxicity model. The objective of this manuscript was to verify the detrimental effect of NAC and its potential mechanism and assess whether 4MP has the same liability. C57BL/6J mice were treated with 300 mg/kg APAP; 9 h after APAP and every 12 h after that, the animals received either 100 mg/kg NAC or 184.5 mg/kg 4MP. At 24 or 48 h after APAP, parameters of liver injury, mitochondrial biogenesis and cell proliferation were evaluated. Delayed NAC treatment had no effect on APAP-induced liver injury at 24 h but reduced the decline of plasma ALT activities and prevented the shrinkage of the areas of necrosis at 48 h. This effect correlated with down-regulation of key activators of mitochondrial biogenesis (AMPK, PGC-1α, Nrf1/2, TFAM) and reduced expression of Tom 20 (mitochondrial mass) and PCNA (cell proliferation). In contrast, 4MP attenuated liver injury at 24 h and promoted recovery at 48 h, which correlated with enhanced mitochondrial biogenesis and hepatocyte proliferation. In human hepatocytes, 4MP demonstrated higher efficacy in preventing cell death compared to NAC when treated at 18 h after APAP. Thus, due to the wider treatment window and lack of detrimental effects on recovery, it appears that at least in preclinical models, 4MP is superior to NAC as an antidote against APAP overdose.

Keywords: Acetaminophen; Drug hepatotoxicity; Fomepizole; Mitochondrial biogenesis; N-acetylcysteine; Regeneration.

Figure 1:. Effect of delayed N-acetylcysteine treatment on liver injury.

Mice were treated with 300 mg/kg APAP or saline (10 ml/kg) IP. Then, 9h later, mice received 100 mg/kg NAC IP followed by additional doses every 12h. Blood and liver tissue samples were collected at 24 and 48h post-APAP. (A) Plasma alanine aminotransferase (ALT) activities. (B) H&E-stained liver sections. (C) Area of necrosis at 48 h (%). Bars represent means ± SE for n = 9 mice per group and time point. *P<0.05 vs. APAP.

Figure 2:. Effect of delayed N-acetylcysteine treatment on DNA fragmentation and peroxynitrite formation.

Mice were treated with 300 mg/kg APAP or saline (10 ml/kg) IP. Then, 9h later, mice received 100 mg/kg NAC IP followed by additional doses every 12h. (A) TUNEL-stained liver sections (24, 48h). (B) Nitrotyrosine-stained liver sections (24h).

Figure 3:. Effect of delayed N-acetylcysteine treatment on regeneration and mitochondrial mass.

Mice were treated with 300 mg/kg APAP or saline (10 ml/kg) IP. Then, 9h later, mice received 100 mg/kg NAC IP followed by additional doses every 12h. Representative images of immunofluorescence staining for PCNA (A) and Tom 20 (B) of tissue sections collected at 24 and 48h.

Figure 4:. Effect of delayed N-acetylcysteine treatment on AMP-activated protein kinase activation and GSH levels.

Mice were treated with 300 mg/kg APAP or saline (10 ml/kg) IP. Then, 9h later, mice received 100 mg/kg NAC IP followed by additional doses every 12h. Liver samples were collected 24 and 48h post-APAP. (A) Western blot analysis for P-AMPK and AMPK of APAP and APAP+NAC-treated animals. (B) Densitometry of P-AMPK and AMPK western blots. (C) Fluorescence staining for P-AMPK at 48h. (D) Hepatic GSH levels at 24 and 48h. Bars represent means ± SE for n = 3 mice per group and time point. *P<0.05 vs APAP.

Figure 5:. Effect of delayed N-acetylcysteine treatment on Nrf2 activation and mitochondrial biogenesis.

Mice were treated with 300 mg/kg APAP or saline (10 ml/kg) IP. Then, 9h later, mice received 100 mg/kg NAC IP followed by additional doses every 12h. Liver samples were collected 48 h post-APAP. (A) Western blot analysis for NRF1, NRF2, Keap1, and β-Actin. Fluorescence staining for PGC-1α (B) and TFAM (C) at 48h after APAP.

Figure 6:. Effect of delayed 4-methylpyrazole treatment on liver injury.

Mice were treated with 300 mg/kg APAP or saline (10 ml/kg) IP. Then, 9h later, mice received an initial bolus dose of 184.5 mg/kg 4MP (HED of 15 mg/kg) IP followed by maintenance doses of 123 mg/kg 4MP (HED of 10mg/kg) every 12h. Blood and liver tissue were collected at 48h post-APAP. (A) Plasma alanine aminotransferase (ALT) activities after APAP treatment; (B) H&E staining of liver tissue sections. Bars represent means ± SE for n = 6 mice per group. *P<0.05 vs. APAP.

Figure 7:. Effect of delayed 4-methylpyrazole treatment on liver recovery.

Mice were treated with 300 mg/kg APAP or saline (10 ml/kg) IP. Then, 9h later, mice received an initial bolus dose of 184.5 mg/kg 4MP (HED of 15 mg/kg) IP followed by maintenance doses of 123 mg/kg 4MP (HED of 10mg/kg) every 12h. (A) Representative images of immunofluorescence staining for PCNA, Tom 20 and P-AMPK of liver sections collected at 24 and 48h post-APAP. (B) Quantitation of fluorescence signals. Bars represent means ± SE of 3 animals per group. *P<0.05 vs APAP.

Figure 8:. Effect of delayed N-acetylcysteine and 4-methylpyrazole treatment on cell death in human hepatocytes.

Freshly isolated primary human hepatocytes were treated with 10 mM APAP. At 18h after APAP, 10 mM or 20 mM 4MP or NAC were added, and cell death was evaluated at 48h. (A) Propidium iodide, phase contrast and merged images of control and treated cells. (B) % of cell death based on ALT release. Bars represent means ± SE for 3 separate cell isolations. *P<0.05 versus Control; ^#P< 0.05 versus APAP.