Mitochondrial protein adducts formation and mitochondrial dysfunction during N-acetyl-m-aminophenol (AMAP)-induced hepatotoxicity in primary human hepatocytes

Abstract

3'-Hydroxyacetanilide orN-acetyl-meta-aminophenol (AMAP) is generally regarded as a non-hepatotoxic analog of acetaminophen (APAP). Previous studies demonstrated the absence of toxicity after AMAP in mice, hamsters, primary mouse hepatocytes and several cell lines. In contrast, experiments with liver slices suggested that it may be toxic to human hepatocytes; however, the mechanism of toxicity is unclear. To explore this,we treated primary human hepatocytes (PHH) with AMAP or APAP for up to 48 h and measured several parameters to assess metabolism and injury. Although less toxic than APAP, AMAP dose-dependently triggered cell death in PHH as indicated by alanine aminotransferase (ALT) release and propidium iodide (PI) staining. Similar to APAP, AMAP also significantly depleted glutathione (GSH) in PHH and caused mitochondrial damage as indicated by glutamate dehydrogenase (GDH) release and the JC-1 assay. However, unlike APAP, AMAP treatment did not cause relevant c-jun-N-terminal kinase (JNK) activation in the cytosol or phospho-JNK translocation to mitochondria. To compare, AMAP toxicity was assessed in primary mouse hepatocytes (PMH). No cytotoxicity was observed as indicated by the lack of lactate dehydrogenase release and no PI staining. Furthermore, there was no GSH depletion or mitochondrial dysfunction after AMAP treatment in PMH. Immunoblotting for arylated proteins suggested that AMAP treatment caused extensive mitochondrial protein adduct formation in PHH but not in PMH. In conclusion, AMAP is hepatotoxic in PHH and the mechanism involves the formation of mitochondrial protein adducts and mitochondrial dysfunction.

Keywords: 3′-Hydroxyacetanilide (AMAP); Acetaminophen; Hepatotoxicity; Mitochondrial dysfunction; Protein adducts.

Figure 1. AMAP induces cell death and GSH depletion in human hepatocytes

Primary human hepatocytes (PHH) were treated with 10 mM of AMAP or APAP over a period of 48 h, and the percentage of alanine aminotransferase (ALT) release was measured at 24 h (A) and 48 h (B) to evaluate toxicity. (C) Dose-response of AMAP toxicity at 48 h after treatment. (D) Glutathione depletion after 10 mM AMAP or APAP exposure for 24 h. Data represent mean ± SE from experiments using cells from 3 to 10 donors. *P< 0.05 (compared with controls); ^#P< 0.05 (compared with APAP group).

Figure 2. AMAP and APAP induce necrosis in human hepatocytes

Primary human hepatocytes (PHH) were treated with 10 mM of AMAP or APAP over a period of 48 h and necrotic cell death as indicated by nuclear PI staining was assessed. DAPI, 4′,6-Diamidino-2-Phenylindole (nuclear stain); PI, propidium iodide (nuclear stain of necrotic cells)

Figure 3. AMAP triggers mitochondrial dysfunction in PHH

(A) Percentage of glutamate dehydrogenase (GDH) release into the culture medium at 48 h after 10 mM AMAP or APAP. (B) Dose-response of GDH release at 48 h after AMAP (5–20 mM). (C) Loss of mitochondria membrane potential after 24 h exposure to 10 mM AMAP or APAP as indicated by the decrease of the red/green fluorescence ratio using the JC-1assay. Data represent mean ± SE from experiments using cells from 8 donors. *P< 0.05 (compared with controls); ^#P< 0.05 (compared with APAP group).

Figure 4. No JNK activation or mitochondrial translocation after AMAP in PHH

JNK activation in the cytosol (A) and P-JNK translocation to the mitochondria (B) were evaluated by western blotting. Mitochondrial fraction from APAP-treated PHH was used as positive control. (C) Densitometry of cytosolic JNK activation. Data represent mean ± SE from densitometry using cells from 3 donors.

Figure 5. No significant mitochondrial dysfunction or cell death after AMAP in primary mouse hepatocytes (PMH)

(A) Lactate dehydrogenase was measured as an indicator of cell death after exposure to 5–20 mM AMAP or 5 mM APAP for 15 h. (B) GSH depletion at 5 h after 5 mM AMAP or APAP. Loss of mitochondrial membrane potential at 5 h (C) and 16 h (D) after AMAP and APAP were assessed by decrease of red/green fluorescence ratio using the JC-1assay. Data represent mean ± SE from experiments using primary mouse hepatocytes from 4 different isolations. *P< 0.05 (compared with controls); ^#P<0.05 (compared with the APAP group).

Figure 6. APAP but not AMAP caused cell necrosis in mouse hepatocytes

Primary mouse hepatocytes (PMH) were treated with 5 mM of AMAP or APAP over a period of 15 h and necrotic cell death as indicated by nuclear PI staining was assessed. DAPI, 4′,6-Diamidino-2-Phenylindole (nuclear stain); PI, propidium iodide (nuclear stain of necrotic cells)

Figure 7. AMAP induces covalent protein binding in PHH but not PMH

(A) Mitochondrial fractions were isolated from PHH and PMH which were treated with AMAP for 6 h or 24h. Mitochondrial fraction from APAP-treated PMH was used as a positive control, and a blank (bl) lane loaded with only SDS loading buffer was used as a negative control. 0 h lanes represent mitochondria samples of control PHH or control PMH without any treatment. Porin was used as a loading control. (B) (C) Densitometry of adduct levels normalized to porin for PHH and PMH. The results of each 0 h lane were set to 1 and the data for AMAP-treated cells are expressed as fold increase over baseline. Data represent mean ± SE from experiments using primary human or mouse hepatocytes from 3 different isolations per group. *P< 0.05 (compared with corresponding controls)