AILI is a prominent cause of ALF in the US; it can primarily be attributed to unintentional as well as intentional ingestion of above therapeutic doses . However, 7% of acute liver failures are at therapeutic doses, as reported by the acute liver failure group, who determined the incidence, risk factors, and outcomes of APAP-induced ALF at 22 tertiary care centers in the United States. As APAP serum concentrations in cases of ALF at therapeutic APAP doses were similar to those in overdose patients and the median APAP concentration differed by only a factor of two (14. 5 μg/dL versus 31 μg/dL), one might possibly suggest self-reported doses and time of ingestion to be unreliable. The seminal study of Larson et al.  reported a dramatic increase in APAP-induced ALF particularly in patients with chronic pain and co-medications (narcotics and/or depressants) as well as alcohol abuse. Thus, identifying biomarkers that would predict individuals at risk for AILI prior to drug treatment would greatly improve drug safety. For this purpose the serum proteome of healthy subjects was analyzed prior to and after treatment with the maximum acceptable dose for APAP given in quarterly portions per day for 7 days. This identified important pre-treatment serum protein associations in individuals that subsequently developed liver injury upon drug treatment.
Regulation of acute phase proteins in AILI responders
A significant difference in expression of serum markers of inflammation was observed in AILI responders versus non-responders. Of the various positive and negative acute phase reactants, CRP, haptoglobin, ApoA-I, beta-2 glycoprotein 1 (ApoH) and RBP are important pre-treatment associations. The mean CRP in 3 out of 12 DILI susceptible subjects was nearly 3-fold higher than the normal upper limit, possibly indicating a mild pre-existing inflammation even though active inflammation of the liver would be associated with CRP levels well above 100 mg/L. Nonetheless, all subjects entering the study were vigorously tested and ALT as well as all other laboratory parameters were within normal ranges. While pre-dose CRP serum concentrations were significantly elevated in ALT responders, its expression was basically unchanged after 7 days of treatment. Notably, CRP serum concentrations and peak ALT measurements appear to correlate (Additional file 4). It is of considerable importance that CRP stimulates phagocytosis of Kupffer cells but decreases their TNF production in a complex manner in which various Fc receptors are involved .
A further biomarker for DILI susceptibility is haptoglobin. This positive acute phase reactant functions as a protective anti-oxidant  and plays a role in the reticuloendothelial system. This system is composed of monocytes and macrophages and is part of the immune system to remove cell debris as observed in cytolitic hepatitis. Haptoglobin binds hemoglobin and plays a crucial role in heme iron recovery to prevent its loss and to protect kidneys from damage by renal iron loading . Prior to APAP treatment, haptoglobin isoforms with different pI but similar Mr (<20 kDa) were significantly (P < 0.01) up-regulated in ALT responders (Figures 3C and 4). Of note, the isoforms remained significantly up-regulated after repeated APAP treatment for 7 days. Diverse functions have been attributed to this tetrameric glycoprotein, with recent evidence pointing to a function as a chemo-attractant for macrophages .
Haptoglobin deficiency is associated with attenuation of hepatosteatosis and impairment of glucose homeostasis, suggesting this protein has a wider role in liver injury. Because of haptoglobin’s role in iron metabolism, the expression of hemopexin was studied as well but remained unchanged in all of the study groups investigated (Additional file 7).
Additional markers of inflammation were significantly regulated and included the acute phase reactant A1AT in native and albumin-depleted sera of ALT responders. This serpin is mainly produced by the liver and irreversibly inhibits trypsin, chymotrypsin and plasminogen activator. In DILI patients the serum concentration of alpha-1-antitrypsin and activity of the transaminases ALT and aspartate aminotransferase (AST) are significantly correlated ; however, A1AT serum concentrations are reduced in ALF . Infantile liver cirrhosis is linked to carriers of the homozygous allele Z or M-Malton of the A1AT gene  while the Pittsburgh variant causes hemorrhagic diathesis.
Another major acute phase reactant significantly up-regulated in AILI susceptible subjects is serum amyloid A (SAA). During an acute phase response this protein is rapidly synthesized by the liver; its expression is regulated by IL-1, IL-6 and TNFα. SAA is an apolipoprotein (apoSAA) associated with high-density lipoprotein and together with CRP are sensitive markers for inflammation. In DILI patients SAA proteins are significantly regulated; a positive correlation between alkaline phosphatase activity and expression of serum amyloid A2 (isoform a) has been reported .
Conversely, APAP treatment caused significant down-regulation of the negative acute phase reactant transthyretin in AILI responders. This triiodothyroxine binding protein is referred to as pre-albumin and is significantly regulated in inflammation. Transthyretin interacts with RBP and was reported as significantly regulated in DILI  and non-alcoholic fatty liver disease (NAFLD) patients of different types . It was also shown to be initially repressed but subsequently up-regulated by more than six-fold in sera of APAP-treated rats after 6 and 24 h, respectively .
About 40% of plasma transthyretin circulates as a complex with RBP. This protein is another negative acute phase reactant and a member of the lipocalin family; the complex stabilizes the binding of retinol to RBP to decrease its glomerular filtration and renal catabolism. Importantly, RBP was significantly down-regulated in ALT responders amongst APAP-treated healthy volunteers; a similar two-fold down-regulation was reported for APAP-treated rats . Independent studies with DILI and NAFLD patients observed similar regulation of RBP [20, 23].
A further acute phase reactant regulated in albumin-depleted sera of ALT responders is ceruloplasmin. This multicopper oxidase plays an important role in oxygen detoxification and is up-regulated during inflammation and hyperoxia . The enzyme oxidizes Fe2+ to Fe3+ without release of radical oxygen species. It is involved in iron transport across the cell membrane. Ceruloplasmin is synthesized by the liver and secreted into plasma by hepatic stellate cells as well as Kupffer cells . In Morbus Wilson patients ceruloplasmin serum levels are decreased.
Drug-induced liver inflammation and immune response
Of particular interest is ADA, which is expressed in all tissues and in large amounts of T lymphocytes. Expression of this enzyme was significantly up-regulated in native serum of ALT responders after 7 days of APAP treatment. The release of ADA into serum documents cellular damage in response to APAP treatment. An important function of serum ADA is the conversion of adenosine to the nucleoside inosine, which is unable to bind to adenosine receptors, thereby influencing neutrophil degranulation, blood flow and oxygen consumption. It is tempting to speculate that increased serum ADA results in the production of anti-inflammatory molecules similar to those seen with non-selective adenosine receptor antagonists, that is, xanthine derivatives. Lack of ADA causes immunodeficiency and knockout mice die perinatally due to severe liver cell degeneration [27, 28]. ADA deficiency is also associated with hyperbilirubinemia and hepatitis but can be resolved by ADA replacement therapy . In acute infective hepatitis serum ADA levels increase; a positive correlation between serum ADA and total bilirubin was reported . This positive correlation may suggest bilirubin functions as an anti-oxidant .
In DILI responders and after repeated APAP treatment IGHG1 was significantly reduced. Importantly, immunoallergic DILI is associated with idiosyncratic liver injury. While reactive metabolites may function as haptens, the parent drug may also elicit an autoimmune reaction with the production of anti-drug antibodies. Usually, during immunoallergic DILI IgG levels are increased and auto-antibodies against liver-specific and non-liver-specific antigens are detected. Possibly, the observed reduction of IGHG1 in AILI susceptibility is due to glutathione depletion in lymphocytes as suggested by Spielberg and Gordon .
Inflammation and coagulation homeostasis in drug-induced liver injury
The complex interplay between drug-induced inflammation and coagulation homeostasis is the subject of intense research. Synthesized in the liver, plasminogen is converted into plasmin by plasminogen activators and bound to fibrin. Plasmin dissolves fibrin of blood clots and acts as a proteolytic factor in a variety of biological processes, including inflammation. It is activated by the urokinase-type plasminogen activator, collagenases and several complement zymogens, such as C1 and C5. It is inactivated by alpha-2-antiplasmin immediately after dissociation from the clot. Importantly, plasminogen serum concentration was significantly reduced in AILI-susceptible patients after 7 days of APAP treatment. Several reports describe a role of plasminogen in macrophage accumulation during liver repair ; plasminogen appears to be essential for neutrophils to accumulate at the border of damaged liver tissue to facilitate repair events . Treatment of mice with APAP caused activation of the coagulation system and protease-activated receptor signaling to contribute to liver injury . Furthermore, fibrin deposition aggravated liver injury; however, plasminogen deficiency reduced APAP liver injury  as observed in plasminogen-deficient mice and wild-type mice treated with tranexamic acid, an inhibitor of plasminogen activation. In the present study serum plasminogen concentrations of AILI responders were statistically significantly reduced, therefore demonstrating clinical relevance of the animal findings. Equally, serum plasminogen concentrations were reduced in APAP-treated non-ALT responders, reinforcing the notion that plasminogen is involved in the fine-tuning of liver regeneration after AILI.
Significant down-regulation of kininogen-1 in serum of ALT responders was another finding. This protein functions as a thiol protease inhibitor. High molecular weight kininogen (HMWK) plays an important role in blood coagulation and inflammation and functions as a precursor of kinin, which is part of the vasoactive kinin-kallikrein system. Kininogens also inhibit the thrombin- and plasmin-induced aggregation of thrombocytes and are therefore antithrombotic proteins. Specifically, kininogen is a mediator of inflammation and causes increased vascular permeability, stimulation of nociceptors, release of mediators of inflammation (that is, prostaglandins) and excerts cardioprotective effects (directly via bradykinin action, indirectly via endothelium-derived relaxing factor action).
The complex interplay between collagen, high molecular weight kininogen (HMWK), prekallikrein and factor 12 (Hagemann factor) results in a primary scar. Initially synthesized in the liver, prekallikrein is converted to kallikrein by factor 12 and eventually cleaves HMWK to release bradykinin. In a study by Cordova and colleagues  the activities of the Hagemann factor, HMWK and prekallikrein were reduced and dependent on the degree of liver failure. Equally, plasma kallikrein clearance was markedly reduced in APAP intoxicated rat livers . The observed reduction of HMWK in DILI responders is likely the result of APAP poisoning of hepatocytes.
Regulation of serum apolipoproteins in drug-induced liver injury
The emerging role of lipoproteins in inflammation and cellular signaling is the subject of intense research . Serum proteome profiling of healthy volunteers revealed a significant three-fold down-regulation of ApoA-I in ALT responders after repeated APAP treatment. This protein participates in the reverse transport of cholesterol from tissues to the liver, thereby promoting cholesterol efflux. It functions as a cofactor for the lecithin cholesterol acyltransferase and is a major protein of plasma HDL, typically found in chylomicrons.
ApoA1 expression was negatively correlated with total bilirubin in a patient cohort diagnosed with DILI . These investigators also observed significant regulation of ApoA2, A4 and A4 precursor in NAFLD and various degrees of liver fibrosis. In contrast, ApoC-II was significantly up-regulated in albumin-depleted sera of ALT responders. This protein is part of the very low density lipoprotein (VLDL) particle. The association of ApoC2 with plasma chylomicrons, VLDL and HDL is reversible and functions in the secretion and catabolism of triglyceride-rich lipoproteins. Levels of the related ApoC-III were likewise significantly changed in patients diagnosed with DILI as were ApoC1 levels in serum of patients diagnosed with NAFLD .
Furthermore, ApoE expression was more than four-fold up-regulated in DILI responders upon APAP treatment. This lipoprotein mediates binding, internalization and catabolism of lipoprotein particles and serves as a ligand for the ApoE-recognizing receptor of hepatic tissues. Its expression was reported as significantly up-regulated in serum of patients diagnosed with DILI but not in those with non-alcoholic steatohepatitis [20, 23]. Early work had shown transcriptional and posttranscriptional regulation of ApoE, ApoA1 and ApoA2 in liver tissue at various pathophysiological states  and changes in lipoprotein binding and uptake by hepatocytes during rat liver regeneration . In a proteomic investigation of drug-induced steatosis a moderate but statistically significant up-regulation of ApoE was observed in rat liver, further documenting its prognostic value in predicting DILI. Additionally, DeKroon and co-workers reported an anti-apoptotic role of ApoE , while others reported that ApoE protects against severe liver disease in hepatitis C virus infection .
Conversely, ApoH was significantly down-regulated in ALT responders after APAP treatment. This protein is synthesized by the liver and secreted in plasma, associates with heparin and dextran sulfate and may prevent activation of the intrinsic blood coagulation cascade by binding to phospholipids on the surface of damaged cells. ApoH was also significantly regulated in rat serum after APAP exposure .
Deoxyribonuclease-2-alpha protein was uniquely down-regulated in response to APAP treatment. This protein hydrolyzes DNA with preference for double-stranded DNA and plays a major role in the degradation of nuclear DNA in cellular apoptosis during development. Its lysosomal localization and its known role in the degradation of exogenous DNA encountered by phagocytosis suggest an interplay of this protein with Kupffer cells in AILI. The reduced expression of this protein may be part of the fine tuning of liver regeneration in response to APAP treatment.
Finally, in DILI responders sulfide:quinone oxidoreductase was significantly regulated. This mitochondrial enzyme was shown to catalyze the oxidation of hydrogen sulfide and belongs to the SQRD family of proteins . It is tempting to speculate that this protein is involved in the metabolism of glutathione (GSH) or cysteine conjugates of APAP metabolites.
The following caveats need to be considered. First, the study is based on APAP given to healthy individuals and although robust statistical relationships were determined, further studies are required so that the findings can be extrapolated to other types of DILI in terms of molecular mechanisms and clinical features. Second, the drugs tested in hepatocyte cultures are idiosyncratic agents; nonetheless, the mechanisms of liver injury may differ amongst them. While transcript regulation of serum acute phase reactants could be demonstrated for a wide range of drugs and by comparison with published serum proteome profiling in DILI patients, additional studies are needed to confirm the results obtained after APAP treatment.