Acetaminophen cytotoxicity in HepG2 cells is associated with a decoupling of glycolysis from the TCA cycle, loss of NADPH production, and suppression of anabolism

Volker Behrends,Guro F Giskeødegård,Natalia Bravo-Santano,Hector C Keun,Michal Letek

doi:10.1007/s00204-018-2371-0

Abstract

Acetaminophen (APAP) is one of the most commonly used analgesics worldwide, and overdoses are associated with lactic acidosis, hepatocyte toxicity, and acute liver failure due to oxidative stress and mitochondrial dysfunction. Hepatoma cell lines typically lack the CYP450 activity to generate the reactive metabolite of APAP observed in vivo, but are still subject to APAP cytotoxicity. In this study, we employed metabolic profiling and isotope labelling approaches to investigate the metabolic impact of acute exposure to cytotoxic doses of APAP on the widely used HepG2 cell model. We found that APAP exposure leads to limited cellular death and substantial growth inhibition. Metabolically, we observed an up-regulation of glycolysis and lactate production with a concomitant reduction in carbon from glucose entering the pentose-phosphate pathway and the TCA cycle. This was accompanied by a depletion of cellular NADPH and a reduction in the de novo synthesis of fatty acids and the amino acids serine and glycine. These events were not associated with lower reduced glutathione levels and no glutathione conjugates were seen in cell extracts. Co-treatment with a specific inhibitor of the lactate/H+ transporter MCT1, AZD3965, led to increased apoptosis in APAP-treated cells, suggesting that lactate accumulation could be a cause of cell death in this model. In conclusion, we show that APAP toxicity in HepG2 cells is largely independent of oxidative stress, and is linked instead to a decoupling of glycolysis from the TCA cycle, lactic acidosis, reduced NADPH production, and subsequent suppression of the anabolic pathways required for rapid growth.

Highlights

Acetaminophen (APAP, paracetamol, or N-acetyl-p-aminophenol) is one of the world’s most commonly used drugs due to its antipyretic and analgesic properties. While it is widely used and generally safe to take at low dosage, APAP is the world’s leading cause of drug overdose and acute liver failure, with a number of these episodes being attributed to unintentional overdosing on APAP/opioid combination analgesics (Bunchorntavakul and Reddy 2013; Yoon et al 2016)
As previously reported (Manov et al 2002, 2004; Macanas-Pirard et al 2004), APAP is toxic and results in cell death in HepG2 cells with toxicity showing a clear concentration dependence and a clear drop in cell viability is visible in the low mM range (24 h exposure, Fig. 1a, Fig. S1)
The question remains: what are the means by which APAP causes toxicity in this cell line, and what is the role of metabolic processes in this toxicity? The data presented here provide evidence in favour of altered mitochondrial metabolism and suppression of anabolism being major consequences of toxic doses of APAP in HepG2 cells, and that resulting lactic acidosis may be contributing to cell death

Summary

Introduction

Acetaminophen (APAP, paracetamol, or N-acetyl-p-aminophenol) is one of the world’s most commonly used drugs due to its antipyretic and analgesic properties. While it is widely used and generally safe to take at low dosage (the FDA-approved dose is less than 4 g per person per day), APAP is the world’s leading cause of drug overdose and acute liver failure, with a number of these episodes being attributed to unintentional overdosing on APAP/opioid combination analgesics (Bunchorntavakul and Reddy 2013; Yoon et al 2016). About 50–70% of APAP is glucoronidated [mainly via UGT1A1 and/ or UGT1A6 (McGill and Jaeschke 2013)] and 25–35% is. The remaining 5–15% are oxidised to the highly reactive N-acetyl-p-benzoquinone imine (NAPQI) by the P450 enzymes, mainly CYP2E1 (Dai and Cederbaum 1995)

Methods

Results

Conclusion