Adaptation to acetaminophen exposure elicits major changes in expression and distribution of the hepatic proteome

Acetaminophen (paracetamol, APAP) overdose is the leading cause of acute liver failure in the USA and UK, resulting in over 600 deaths a year in these countries1,2. Whilst a single dose of 10–15 g is likely to result in severe liver damage3, prolonged exposure to acetaminophen has been shown to result in autoprotection in some patients, such that daily doses even in excess of 10 g have apparently little adverse effect. In one extreme example, APAP-induced autoprotection was demonstrated in an adult male addicted to the analgesic Percocet (APAP formulated with oxycodone), who consumed up to 65 g per day of APAP4. In addition, volunteers administered a daily therapeutic dose (4 g) of APAP displayed elevations in circulating liver enzymes (clinical markers of liver injury), which then resolved5. Autoprotection is therefore likely to be an important human defensive mechanism to prevent progressive injury resulting from drug toxicity. Autoprotection to APAP has also been recapitulated in animal models: in mice, daily escalating doses can tolerize against liver damage within a week of treatment4. Our knowledge of the mechanism of hepatic adaptation is limited, and focus to date has been on select proteins implicated in APAP toxicity. APAP liver damage is caused by a metabolite – N-acetyl-p-benzoquinoneimine (NAPQI) – thus enzymes involved in the formation or detoxification of NAPQI are likely to be involved in the adaptive response. In particular, cytochrome P450 (CYP) 2E1 which activates APAP to NAPQI and the multidrug resistance-associated proteins ABCC3 and ABCC4 (also known as MRP3 and MRP4), which transport APAP and other xenobiotics out of cells, have been implicated in the autoprotection4,6. Alternatively, proteins involved in the cell’s natural defence systems, including those regulating glutathione (GSH), may underlie the adaptation. A recent microarray study also linked the expression of a number of novel genes to the development of tolerance to APAP7. Induction of flavin-containing monooxygenase-3 (FMO3), an enzyme identified in this study that has not previously been associated with APAP metabolism, was subsequently shown to be protective in an APAP autoprotection model8. Here, using a rat model, we have investigated this process and show that in fact the expression of as many as 30% of all proteins detected in the liver is altered during adaptation to APAP, and see a dramatic shift in the localisation of CYP2E1. This indicates that the process of adaptation to APAP-induced liver injury is more extensive and dynamic than previously thought.