Mechanisms of acetaminophen-induced cell death in primary human hepatocytes

Abstract

Acetaminophen (APAP) overdose is the most prevalent cause of drug-induced liver injury in western countries. Numerous studies have been conducted to investigate the mechanisms of injury after APAP overdose in various animal models; however, the importance of these mechanisms for humans remains unclear. Here we investigated APAP hepatotoxicity using freshly isolated primary human hepatocytes (PHH) from either donor livers or liver resections. PHH were exposed to 5mM, 10mM or 20mM APAP over a period of 48 h and multiple parameters were assessed. APAP dose-dependently induced significant hepatocyte necrosis starting from 24h, which correlated with the clinical onset of human liver injury after APAP overdose. Interestingly, cellular glutathione was depleted rapidly during the first 3h. APAP also resulted in early formation of APAP-protein adducts (measured in whole cell lysate and in mitochondria) and mitochondrial dysfunction, indicated by the loss of mitochondrial membrane potential after 12h. Furthermore, APAP time-dependently triggered c-Jun N-terminal kinase (JNK) activation in the cytosol and translocation of phospho-JNK to the mitochondria. Both co-treatment and post-treatment (3h) with the JNK inhibitor SP600125 reduced JNK activation and significantly attenuated cell death at 24h and 48h after APAP. The clinical antidote N-acetylcysteine offered almost complete protection even if administered 6h after APAP and a partial protection when given at 15 h.

Conclusion: These data highlight important mechanistic events in APAP toxicity in PHH and indicate a critical role of JNK in the progression of injury after APAP in humans. The JNK pathway may represent a therapeutic target in the clinic.

Keywords: Acetaminophen protein adducts; Drug-induced liver injury; Mitochondrial dysfunction; Oncotic necrosis; c-Jun-N-terminal kinase.

Figure 1. APAP induces cell death in primary human hepatocytes (PHH)

Cells were treated with 5mM or 10mM APAP over a period of 48 hours. (A) Time course of toxicity over 48 hours, as indicated by percentage of alanine aminotransferase (ALT) activity found in the culture medium, (B) Dose-response of APAP toxicity at 24 hours after treatment. (C) Necrotic cell death as indicated by nuclear PI staining after 24h after 10mM APAP exposure. The nucleus was stained with DAPI. Data in A and B represent mean ± SE of 20 independent cell isolations. *P < 0.05 (compared with time 0 or control).

Figure 2. Inter-individual variation of ALT and GSH levels in primary human hepatocytes

(A) ALT release from hepatocytes isolated from 44 patients. Each batch of cells (individual patient) were either untreated (0), or exposed to 10mM APAP for 24 and 48 hours. (B) ALT release from hepatocytes isolated from male and female patients 24 and 48 hours after 10mM APAP. (C) GSH levels of hepatocytes from each individual (n=7) at 0 and 6 hours after 10mM APAP. Horizontal lines indicate average values.

Figure 3. APAP triggers GSH depletion, mitochondria dysfunction and protein adduct formation in PHH

(A) Time course of cellular glutathione depletion after 10mM APAP. (B) Loss of mitochondria membrane potential over 24 hours after 10mM APAP, as indicated by decrease of red/green fluorescence ratio using the JC-1 assay. APAP-protein adduct formation (C) in the whole cell and (D) in the mitochondria fraction over 15 hours after 10mM APAP. Data represent mean ± SE from experiments using cells from 3–8 donors. * P < 0.05 (compared with time 0).

Figure 4. APAP leads to cellular JNK activation in PHH and phospho-JNK translocation to the mitochondria

(A) Time course of JNK activation in whole cell homogenate after 10 mM APAP and (B) Densitometry. (C) JNK activation in the cytosol and phospho-JNK translocation to the mitochondria and (D) Densitometry. Data represent mean ± SE of independent western blotting using PHH samples from 3–4 donors. * P < 0.05 (compared with controls).

Figure 5. JNK inhibitor SP600125 partially protects against APAP-induced cell death in PHH

JNK inhibitor SP600125 using 0.2% DMSO as the vehicle was administered either as (A) a co-treatment with APAP or (B) a 3-hour post-treatment after APAP. (C) Western blotting was performed to verify the efficacy of SP600125. Data represent mean ± SE from experiments using cells from 6 donors. ^#P < 0.05 (compared with APAP+Veh group).

Figure 6. No JNK activation in HepaRG cells after APAP and no protection using JNK inhibitor SP600125

(A) Time course of JNK activation over 24 hours in HepaRG cells. (B) Cell death as suggested by percentage of lactate dehydrogenase (LDH) released into the culture media at 24 hours after APAP with or without JNK inhibitor SP600125 as a co-treatment. Data represent mean ± SE of n=4. * P < 0.05 (compared with control), ^#P<0.05 (compared with APAP group)

Figure 7. Protection by N-acetylcysteine (NAC) against APAP-induced hepatotoxicity in PHH

20mM NAC was applied at various time points after APAP and (A) ALT activity, (B) Glutamate dehydrogenase (GDH) activity and (C–D) JNK activation was evaluated at 24 hours or 48 hours after APAP. (D) Densitometry. Data represent mean ± SE from experiments using cells from 3–7 donors. * P < 0.05 (compared with control), ^#P<0.05 (compared with APAP group)