Abstract
Objective: Acetaminophen (APAP) overdose contributes to liver damage through modulation of pro-apoptotic processing. This study evaluated the involvement of caspase/Bax factors in APAP hepatotoxicity in vivo and in vitro.
 Methods: The involvement of caspase/Bax factors in APAP hepatotoxicity was evaluated in BALB/c mice and on isolated primary mouse hepatocytes. In vitro MTT assay was carried out on primary cultured mouse hepatocytes treated with APAP (2.5, 5, 10 mmol) and Annexin V/PI staining was employed to cell suspension for imaging under fluorescence microscopy. In addition, caspase-3 concentrations were determined in cell lysates. In vivo, mice were treated with a toxic dose of APAP (700 mg/kg) and immunodetection of Bax was made by Western Blot. Vitamin C (Vit C) was used as a hepato-protectant due to its known antioxidant activities.
 Results: In vitro dose-dependent increase in mitochondrial electron transport capacity was evident in isolated mouse primary hepatocytes incubated with the high dose of APAP (10 mmol) compared to both nontreated cells and cells pre-treated with Vitamin C (Vit C) (0.5 mmol) (p<0.05). Apoptosis was confirmed in hepatocytes through Annexin V staining after APAP treatment and the signal was reduced when hepatocytes were pre-treated with Vit C. In addition, caspase-3 concentration was decreased in cells pretreated with Vit C prior to APAP exposure. In vivo, Bax immunodetection utilizing western blotting was increased in mice treated with the toxic dose of APAP (700 mg/kg) and attenuated through pre-treatment with Vit C.
 Conclusion: Modulation of apoptotic caspase/Bax pathway is present in hepatocytes undergoing APAP-induced toxicity.
Highlights
The exact mechanism of toxicity behind Acetaminophen (APAP, known as N-acetyl-para-aminophenol or paracetamol) is not fully elucidated despite its popular worldwide use as a safe and effective analgesic and antipyretic at therapeutic doses
It has been reported that APAP is capable of inducing hepatotoxicity in rats in a similar manner to that induced in mice when a three-fold higher dose is employed [17, 18]
The mitochondrial apoptotic pathway has been considered as a vital mean of initiating cell survival [19], as it initiates the production of pro-apoptotic proteins in response to the induced insult, activating caspase activity and apoptosis [20]
Summary
The exact mechanism of toxicity behind Acetaminophen (APAP, known as N-acetyl-para-aminophenol or paracetamol) is not fully elucidated despite its popular worldwide use as a safe and effective analgesic and antipyretic at therapeutic doses. Overdosing with APAP results in a fully saturated conjugative pathway shifting APAP metabolism into the cytochrome P450 (CYP450) pathway, leading to increased production of the toxic metabolite, namely, Nacetyl-p-benzoquinone imine (NAPQI), which reduces glutathione (GSH) levels until depleted allowing disruptive binding to mitochondrial proteins [2]. Before NAPQI manages to induce cell death, initial mitochondrial metabolic activation has been reported to be critical as it needs to be amplified and propagated in order to initiate the cellular injury [4]. The integrity of mitochondrial architecture is lost causing depletion of ATP and translocation of Bax, a pro-apoptotic Bcl-2 protein, from the cytosol to the mitochondrion that causes pore formation in the outer mitochondrial membrane. Once the cell undergoes apoptosis, an inner leaflet restricted lipid component called phosphatidylserine (PS) is translocated to the outer membrane scrambling the plasma membrane lipid asymmetry, indicating apoptosis [7]
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