2P caspases mediate the execution of apoptosis by paracetamol (acetaminophen) in human HuH7 hepatablastoma cells A Patricia Macanas-Pirard, 1Pauline C. Lee, Richard H. Hinton, Nick J. Toms and George E.N. Kass. School of Biomedical & Life Sciences, University of Surrey, Guildford, Surrey GU2 7XH and 1GlaxoSmithKline Research and Development, Ware, Herts SG12 0DP, UK.

Print abstract Search PubMed for: Macanas AP Lee PC Hinton RH Toms NJ Kass EN

The widely used analgesic drug paracetamol (acetaminophen, AAP) can induce fatal liver injury through a combination of apoptosis and necrosis, when taken in large doses. We have previously shown that apoptosis plays a critical role in initiating AAP-induced hepatic injury since inhibiting apoptosis also prevents the development of acute liver failure (El-Hassan et al. submitted). In this study, the mechanism of AAP-induced apoptosis was investigated in vitro employing the human hepatoblastoma cell line, HuH7.

HuH7 cells were grown in DMEM supplemented with 10 % foetal bovine serum. Chromatin morphology was assessed with the fluorescent nucleic acid stain bisbenzimide H 33258 (3 µg ml^-1) and DNA fragmentation quantified by flow cytometric analysis of propidium iodide (10 µg ml^-1)-stained cells (Jones et al., 1998; Coley et al., 1999). Cytotoxicity was determined via monitoring lactate dehydrogenase (LDH) release (Roche). Mitochondrial membrane potential (m) was examined using the mitochondrial fluorophore tetramethylrhodamine ethyl ester (TMRE, 100 nM) (Gross and Loew, 1989). Cytosolic cytochrome c translocation was examined via indirect immunofluorescence (mouse anti-cytochrome c monoclonal antibody (mAb) (1:100, BD Pharmingen) / FITC-labelled anti-mouse secondary polyclonal antibody (pAb) (1:100, DAKO)). Caspase processing was qualitatively examined by Western blot (WB) analysis (anti-caspase-3 (Calbiochem) and anti-caspase-7 pAbs) and quantitatively by flow cytometric analysis using an anti-active caspase-3 mAb (1:10, BD Pharmingen, phycoerythrin conjugated.

Where appropriate the pan-caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp fluoromethylketone (Z-VAD-fmk; 100µM) was incubated for 30 min prior to AAP addition. Flow cytometric immunodetection of cleaved cytokeratin-18 (M30 mAb, Roche) was carried out to measure in situ caspase-3 activation. Data are expressed as mean (± S.D.) and represent a 48 hr exposure duration with 10 mM AAP, unless otherwise stated. Statistical analyses were performed using the unpaired Student's t-test.

When tested at 10 mM, AAP elicited significant cytotoxicity (AAP = 62 ± 7 % and control = 3 ± 3 % LDH release, n=3, at 54 h, P<0.001) and accompanying DNA fragmentation (AAP = 53 ± 8%, control = 4 ± 1%, n=3, P<0.01). Processing of caspase-3 and caspase-7 to their corresponding active fragments (p17/p20 and p19) was detected by WB. Caspase-3 activation was quantitated by flow cytometry (AAP = 43 ± 2%, control = 4 ± 1% gated, n=3, P<0.001) which correlated with cytokeratin-18 cleavage (AAP = 40 ± 1 % and control = 3 ± 2 % gated, n=3, P<0.001). The manifestation of apoptosis was preceded by decreased mitochondrial TMRE fluorescence (hence m loss) and diffuse cytosolic cytochrome c immunoreactivity. Z-VAD-fmk afforded significant protection from AAP-induced cell death (AAP = 80 ± 10 % and AAP + Z-VAD-fmk = 8 ± 1 % LDH release, n=3, at 54h) supporting a role of caspases in AAP-induced apoptosis and subsequent cell death.

In conclusion, AAP induces apoptosis in human hepatoblastoma HuH7 cells through mitochondrial membrane depolarisation, mitochondrial cytochrome c release and caspase activation.

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