In this study, two dimensional gel electrophoresis and mass spectrometry were used to profile serum proteins during acute liver injury and recovery in rats after acetaminophen exposure. More than 800 densiometric features were reproducibly detected on each two dimensional gel for each animal. Serum proteins as measured by differential two dimensional gel electrophoresis appeared remarkably stable in the absence of liver injury since so few changes were observed from 6–48 hr at the lower dose of acetaminophen or at 6 hr after an injury producing dose. However, during the time of peak toxicity with a known liver injury dose of acetaminophen as determined by histopathology and ALT/AST activities, differential proteomic analysis showed the greatest number of serum protein changes. Of the 68 gel features that were altered, almost 80% were identified by mass spectrometry. Many of these altered serum proteins in acetaminophen-induced liver injury have functions in acute phase response, coagulation, scavenging, transport, intermediary metabolism, catabolism or structural functions. By 48 hr, the number of serum protein changes were reduced by four fold compared to those observed at 24hr, clinical serum chemistries had declined, and histologic features in the hepatic centrilobular region indicated regenerative processes were well underway.
There have been only limited proteomic studies of acetaminophen liver injury in rats despite the large body of literature in this species (Park et al., 2005
). Liver and serum were probed by two dimensional electrophoresis in Sprague Dawley rats for new markers of hepatomegaly, hepatic necrosis or hepatobiliary injury after single exposures to four compounds including phenobarbital and Wyeth-14,643 (pirinixic acid), acetaminophen and ANIT, respectively (Amacher et al., 2005
). Similar to the current study, they noted that 1000 mg/kg acetaminophen increased serum 4-HPPD and of 19 altered serum proteins, these four agents increased Gc globulin and malic dehydrogenase and also decreased PRBP. A related proteomic study similarly examining various hepatotoxicants identified 100 to 200 rat liver proteins including reduced levels of hepatic catalase as a common finding (Thome-Kromer et al., 2003
) although this observation remains unexplained.
A primary interest in serum proteomic analysis during acetaminophen hepatotoxicity is to increase understanding of drug-induced liver injury by detection of serum protein changes. A progression of critical events starting from reactive intermediate formation, protein adducts, alterations in hepatocellular gene expression, biochemistry and function eventually impacts the serum proteome. Genomic analysis has shown transcript changes are detectable at early times (15 min) (Ruepp et al., 2002
) or sub-histological toxicity (Heinloth et al., 2004
) although the complexity of cellular events often obscures precise mechanisms of toxicity. In our study, proteomic analysis did not reveal early changes in the serum proteome despite indications that apparent critical events were occurring at 6 hr (i.e. TNFα) prior to the later appearances of elevated serum ALT, AST and necrosis. The detection of early predictors of liver injury in serum await more sensitive proteomic methodologies matched to the kinetic unfolding of critical events in liver injury. However, we did discover the appearance of several serum proteins during peak injury and recovery periods not previously reported. Increases in serum fetuin B isoforms and arginosuccinate synthase, decrease in AT-III and alterations in complement C3 isoforms were consistently observed at 24 and 48 hr. We found serum protein changes previously observed with other drug-induced necroses from hepatic cytoplasmic compartments including aminopeptidase (Musana et al., 2004
), glutamate dehydrogenase (O'Brien et al., 2002
), the urea cycle enzymes arginosuccinate synthase and ornithine carbamoyltransferase (OCT) (Ikemoto et al., 2001
), malic enzyme (Zieve et al., 1985
) and urocanase (Trip et al., 1973
). Increased serum glycogen phosphorylase has been proposed as a marker of acute ischemic cardiovascular disease (Apple et al., 2005
) but is now reported for acetaminophen toxicity.
The findings of increased serum proteins not normally associated with acetaminophen necrosis might also be interpreted as beneficial host responses for recovery from liver injury. Gc-globulin and hemopexin can respectively scavenge free actin and heme released from necrotic cells that might otherwise lead to multiple organ failure after acute liver damage (Lee and Galbraith, 1992
; Tolosano and Altruda, 2002
). The 2 to 6 fold increase in various isoforms of serum complement C3 and C4a may contribute to inflammation and liver regenerative processes after injury (Markiewski et al., 2004
). Increased serum isoforms of the cystatin-like protein, fetuin-B, at 24 and 48 hr are consistent with elevated serum fetuin-B recently reported for a murine leukemia model (Bhat et al., 2005
In the present study, two isoforms of catalase were increased by 10 fold in serum at 24 hr after acetaminophen toxicity. Validation of serum catalase by western blot and biochemistry at 24, 48 and 72 hr demonstrated that it was enzymatically most active at 24–48 hr at times corresponding to greatest liver injury. However, catalase was still present in serum at 72 hr during recovery. The potential for oxidative vascular damage upon endothelia that line the liver (Laskin, 1996
), lung (Hammerschmidt and Wahn, 2004
) brain (Li et al., 2003
) and other organs has been documented by in vitro experiments with hydrogen peroxide. Prior studies report that cultured endothelia are better protected from hydrogen peroxide by the serum of normal human subjects that contain increasing amounts of catalase activity (Leff et al., 1991
). Importantly, therapeutics with catalase-activity have been shown to prevent sinusoidal and hepatocellular damage in models of liver injury (Yabe et al., 1999
). We interpret increased serum catalase during acetaminophen toxicity as a beneficial host response for protecting vascular endothelia from oxidative injury that occurs as an indirect consequence of liver necrosis.
The actions of cytokines, inflammatory mediators and reactive oxidant species have gained increasing prominence for their roles in acetaminophen-induced liver injury (Dambach et al., 2005
; James et al., 2005b
). Acute phase response proteins that we found in serum suggested we search for other serum cytokines and chemokines particularly since these are typically below detection limits for staining in two dimensional gels. We complemented our proteomics strategy with cytokine antibody arrays and ELISA’s to survey further serum protein changes. Serum TNFα levels peaked at 24–48 hr after acetaminophen but remained increased above control up to 120 hr. Similarly, antibody array and ELISA data in our study showed a 24–48 hr peak for MCP-1 and TIMP-1 after acetaminophen in a manner that might reflect a coordination of reparative processes. Proteolytic degradation of the extracellular matrix (ECM) is essential for tissue remodeling in liver repair, involving a combination of matrix metalloproteinases (MMPs) and their specific tissue inhibitors or TIMPs (Rudolph et al., 1999
). A major factor in liver regeneration is the release and activation of pro-HGF from ECM via proteolytic cleavage (Liu et al., 1994
). TIMP-1 is thought to play a central role in regulating release and activation of HGF via the TIMP/MMP proteolytic axis that ultimately controls the HGF-mitogenic signaling pathway (Mohammed et al., 2005
). Although the exact roles of inflammation and increased serum proteins such as catalase, MCP-1 and TIMP-1 as contributory or restorative factors in acute liver injury are controversial (Gardner et al., 2003
; Liu et al., 2004
; Jaeschke et al., 2005
), immune involvement could represent a systemic host defense mechanism to organ injury.
The current capabilities of proteomic technologies combined with the wide quantitative range and complexity of serum protein expression often require use of multiple expression platforms as used in this study. Equally important are pre-purification strategies such as immunodepletion to remove the abundant, less informative serum proteins and allow more facile detection of low concentration serum proteins by proteomic methods. In this rat model, we observed relatively few changes in our probing of the serum proteome in the absence of tissue injury at a subtoxic dose of 150 mg/kg from 6–48 hr, and with a toxic dose at 1500 mg/kg acetaminophen at 6 hr. This apparent stability of the serum proteome is in part due to proteomic platform limits in sensitivity but is also consistent with other rodent studies in which liver transcript alterations precede changes in clinical chemistries or cytokines levels at injury producing acetaminophen doses (Dambach et al., 2002
; James et al., 2005a
) or even subtoxic doses (Heinloth et al., 2004
). As proteomic technologies improve and coverage of the rat serum proteome becomes deeper and better defined, it is probable that more subtle changes in low level regulatory proteins will be observed earlier in the damage process as the animal adjusts to liver injury. Many of the reported proteome changes we observed consisted of passively released cellular contents from damaged hepatocytes as well as actively secreted signaling peptides (cytokines and chemokines) from both resident nonparenchymal (Kupffer, stellate, pit cells) and recruited circulating leukocytes. It is also possible that other organs and tissues actively contribute or secrete proteins as new serum constituents in the face of major organ damage since blood comes in contact with all organs and tissues of the host. Complete description of the serum proteome of the rat would advance an understanding of the biology of host response to liver injury in an important preclinical species while providing new markers that might distinguish between pharmacologic effects and injurious effects of therapeutic agents.