RETRACTED ARTICLE: Diet Restriction Inhibits Apoptosis and HMGB1 Oxidation and Promotes Inflammatory Cell Recruitment during Acetaminophen Hepatotoxicity

Abstract

Acetaminophen (APAP) overdose is a major cause of acute liver failure and serves as a paradigm to elucidate mechanisms, predisposing factors and therapeutic interventions. The roles of apoptosis and inflammation during APAP hepatotoxicity remain controversial. We investigated whether fasting of mice for 24 h can inhibit APAP-induced caspase activation and apoptosis through the depletion of basal ATP. We also investigated in fasted mice the critical role played by inhibition of caspase-dependent cysteine 106 oxidation within high mobility group box-1 protein (HMGB1) released by ATP depletion in dying cells as a mechanism of immune activation. In fed mice treated with APAP, necrosis was the dominant form of hepatocyte death. However, apoptosis was also observed, indicated by K18 cleavage, DNA laddering and procaspase-3 processing. In fasted mice treated with APAP, only necrosis was observed. Inflammatory cell recruitment as a consequence of hepatocyte death was observed only in fasted mice treated with APAP or fed mice cotreated with a caspase inhibitor. Hepatic inflammation was also associated with loss in detection of serum oxidized-HMGB1. A significant role of HMGB1 in the induction of inflammation was confirmed with an HMGB1-neutralizing antibody. The differential response between fasted and fed mice was a consequence of a significant reduction in basal hepatic ATP, which prevented caspase processing, rather than glutathione depletion or altered APAP metabolism. Thus, the inhibition of caspase-driven apoptosis and HMGB1 oxidation by ATP depletion from fasting promotes an inflammatory response during drug-induced hepatotoxicity/liver pathology.

Introduction

Drug-induced liver injury (DILI) is a major clinical concern and a leading cause of acute liver failure (ALF) (1). Acetaminophen (APAP) is a widely used analgesic that is safe at therapeutic doses. APAP hepatotoxicity after overdose contributes to a significant proportion of cases of ALF worldwide (2). Biochemical events that initiate hepatotoxicity through reactive metabolite formation and hepatic glutathione (GSH) depletion are well defined (3,4), with centrilobular necrosis being the eventual form of cell death (5). Despite intense research, the cellular events linking metabolic activation to clinical outcome are not understood. A comprehensive understanding of events leading to DILI would improve clinical management and inform the design of therapeutic interventions.

We have recently identified and characterized keratin-18 (K18) and high mobility group box-1 protein (HMGB1) released from dying hepatocytes in a murine model of APAP hepatotoxicity as sensitive mechanism-based biomarkers, and we observed a hepatoprotective role played through induction of hepatocyte apoptosis (6). Conflicting data exist within the literature regarding the occurrence and consequences of APAP-induced hepatocyte apoptosis during overdose in vivo (7,8). Apoptosis and necrosis frequently coexist in pathological conditions of the liver, and the balance of cell death may be dictated by the particular insult. In general, reported investigations that found that apoptosis was not a feature of APAP-induced hepatotoxicity tend to use fasted animal models (6–10). It is plausible that depletion of hepatic ATP, necessary for the induction of caspase activation, cleavage of caspase substrates and execution of apoptosis, could be a consequence of fasting animals before treatment (11,12).

The role of the innate immune response activated through APAP-induced direct hepatocyte death in animal models also remains controversial (13). Many cell types have been implicated in determining the extent of organ injury or regeneration in the liver, such as Kupffer cells (14), neutrophils (15), natural-killer cells and natural-killer cells with T-cell receptor (16). These downstream events involve release of pro- and antiinflammatory mediators, the balance of which may influence individual or interanimal susceptibility and may be dictated by experimental conditions. The precise role of the various cell types and the signaling mechanisms responsible for cell activation and recruitment are yet unknown, but inflammatory mediators such as tumor necrosis factor (TNF)-α (17) and interferon γ (18) have been implicated in increased susceptibility to APAP hepato-toxicity, whereas interleukin (IL)-6 (19) and IL-10 (20) have been implicated in hepatic regeneration and protection after a toxic insult.

HMGB1 is a chromatin-binding protein that has proinflammatory activity. The release of damage-associated molecular pattern (DAMP) molecules by necrotic cells, such as HMGB1, is thought to play a key role in alerting the immune system to dying cells (21,22). HMGB1 cytokine activity is directed through the interaction with Toll-like receptors (TLR) and the receptor for advanced glycation end products (RAGE) on target cells (23–25). The definitive role played by HMGB1 in the pathogenesis of DILI remains to be fully characterized. In our fed mouse model we observed no histological evidence of hepatic innate immune cell infiltration or activation, despite significantly elevated serum levels of HMGB1, but hepatic regeneration was evident 24 hours after APAP treatment (6). DAMPs undergo posttranslation modifications that can have an impact on their biological function. HMGB1 contains three cysteine residues, and recent evidence has highlighted the importance of cysteine 106 (C106) for the proinflammatory properties of HMGB1 (26). Moreover, the oxidation status of C106 within the cytokine domain of HMGB1 has been hypothesized to regulate its stimulatory activity (27). Oxidation of the sulfhydryl group within C106 has been shown to be a caspase-directed process and is important in the induction of immune tolerance by apoptotic cells and the prevention of potentially harmful effects of proinflammatory DAMPs (27).

In continuation from our previous work (6) we have used HMGB1 and K18 as mechanistic tools in the investigation of whether prefasting of mice inhibits APAP-induced caspase activation and apoptosis through depletion of hepatic ATP (11,12,28), and the subsequent inhibition of HMGB1 oxidation permits the circulation of immune-active, reduced HMGB1 released by necrotic cells. These findings offer a mechanistic insight to resolve inconsistencies within the literature with respect to induction of APAP-induced apoptosis and initiation of an inflammatory response. Determination of the degree to which apoptosis contributes to hepatocyte death in APAP overdose and acts as a protective mechanism through inhibition of a potentially damaging inflammatory response by neutralizing DILI-associated DAMPs is essential for the clinical management of the condition and to inform the development of safer drugs.

Materials and Methods

Materials were purchased as described previously (6). HMGB1-neutralizing antibody and control IgY antibody were from Shino-Test Corporation (Tokyo, Japan) and TNF-α and IL-6 enzyme-linked immunosorbent assay (ELISA) kits from R&D systems (Abingdon, UK).

Experimental Animal Treatment

Protocols undertaken in accordance with a license granted under the Animals (Scientific Procedures) Act 1986 and approved by the University of Liverpool ethics committee. Groups of six male CD-1 mice (25–35 g), which either had free access to food and water or were fasted for 24 h with free access to water were included in the study. Animals were administered a single intraperitoneal injection of APAP (530 mg/kg) in 0.9% saline and euthanized 3, 5 or 24 h after treatment. Control animals received either 0.9% saline or solvent control in 0.9% saline as appropriate. The pan-caspase inhibitor Z-VAD.fmk with and without APAP (530 mg/kg) was administered to fed mice as described previously (6). Other groups of mice received diethyl maleate (DEM) (4.7 mmol/kg) or glucose (1000 mg/kg) and glycine (100 mg/kg) based on previously described protocols (29,30). Hepatic DNA laddering, caspase-3 Western blotting, GSH content assessment and histological and immunohistological analyses for active caspase-3 were performed as previously described (6). To determine the role played by circulating HMGB1 released by dying hepatocytes after APAP treatment, groups of fasted mice intravenously received 200 µg of a neutralizing chicken polyclonal antibody to HMGB1 or 200 µg of a control IgY antibody, which has previously been used (31). Antibody treatment was conducted 2 h after APAP administration (530 mg/kg, 24 h).

Hepatotoxicity Assessment

Histological assessment of hepatotoxicity and the immunohistological examination were carried out as previously recorded (6). The degree of hepatocyte loss was scored 0–5 independently by a pathologist (A Kipar) and another author (DJ Antoine), as previously described (6).

Serum Biomarker Analysis

Serum ALT activity determination, HMGB1 or K18 ELISA and mass spectrometry (MS) analysis were carried out as previously described (6). Western analysis of HMGB1 was carried out as previously described after reducing and nonreducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (6). Reduced and oxidized HMGB1 were identified by Western blot, as previously described, by using a rabbit polyclonal anti-HMGB1 antibody, which does not distinguish between posttranslation modifications (27), and confirmed by liquid chromatography-tandem MS (LC-MS/MS) after tryptic digestion and excision from SDS-PAGE gels.

Determination of APAP Irreversible Binding to Murine Hepatic Protein

[¹⁴C]-APAP (5 µCi ¹⁴C-APAP [530 mg/kg]) covalently bound to hepatic protein was measured by exhaustive solvent extraction as previously described and expressed as nanomole equivalent bound/milligrams hepatic protein (32).

Statistical Analysis

All results (excluding those from histological and immunohistological analyses) are expressed as mean ± standard deviation (SD). Values were analyzed for non-normality by using a Shapiro-Wilk test. The unpaired t test was used when normality was indicated, and the Mann-Whitney U test was used for non-parametric data. All calculations were performed by using StatsDirect statistical software; results were considered significant when P < 0.05.

Results

Fasting of Mice Depletes Basal Hepatic ATP and Inhibits APAP-Induced Apoptosis

In control mice fed ad libitum, a variable degree of hepatocellular glycogen, the ATP precursor, was evident. The periodic acid-Schiff (PAS) reaction highlighted primarily centrilobular hepatocyte glycogen accumulation (Figure 1A). In mice fasted for 24 h, only scattered hepatocytes contained glycogen (Figure 1B). Fasting of mice resulted in reduction of basal hepatic ATP levels by 50% (Table 1). The observed depletion of ATP after APAP treatment was more rapid in fasted mice (Figure 2F). In mice with free access to food, centrilobular cell loss was observed 3 h (average score 1.1) and 5 h (average score 1.5) after APAP. At 3 h numerous centrilobular hepatocytes with apoptotic morphology were observed (Figure 1C). Apoptosis was confirmed by the demonstration of cleaved caspase-3 in these cells (Figure 1D). At 5 h, apoptotic cells were still present in small numbers, and when extensive cell loss was observed. At both time points, procaspase-3 processing, DNA laddering and serum K18 fragment elevations confirmed apoptosis as a substantial type of APAP-induced hepatocyte death (Figure 2A–C). Administration of APAP after a 24-h fasting period induced more extensive early centrilobular cell loss (average score 1.6 at 3 h). Evidence of apoptotic hepatocytes was not present at 3 or 5 h (average score 1.4) after APAP (Figure 1E–F), whereas necrotic hepatocytes were identified centrilobularly (Figure 1E). The findings were confirmed by negative results for cleaved procaspase-3 Western blotting (Figure 2A) and absence of hepatic DNA laddering (Figure 2B). Elevated caspase-cleaved K18 levels were detected in the serum of fed APAP-treated mice but not fasted mice (Figure 2C). Instead, increased serum levels of hypoacetylated HMGB1, full-length K18 and alanine aminotransferase (ALT) activity, all serum markers of necrosis, were observed when mice had been fasted before APAP administration (Table 1 and Figure 2E). There was no difference between fed and fasted mice in the levels of [¹⁴C]-APAP irreversible binding to hepatic protein, a marker of APAP bioactivation (Table 1).

Figure 1

Histological determination of the effect of fasting on APAP-induced hepatocyte apoptosis in mice. (A,B) Control mice, PAS reaction. (A) In control mice with free access to food, the vast majority of centrilobular hepatocytes exhibit cytoplasmic glycogen (arrow). (B) Mice fasted for 24 h do not exhibit glycogen within hepatocytes. Bars = 20 µm. (C-F) 3 hours after APAP treatment (530 mg/kg). In animals with free access to food (C,D), centrilobular cell loss was observed (average score 1.1). There were apoptotic cells that could be identified based on their morphology in a hematoxylin and eosin-stained section (arrows, C) and the expression of cleaved caspase-3 (arrows, D). Bars = 10 µm. Animals that had been fasted for 24 h before APAP administration showed more extensive hepatocyte loss (average score 1.6). Necrotic hepatocytes were identified in the hematoxylin and eosin-stained section (arrows, E). There was no evidence of cleaved caspase-3 expression by hepatocytes, and the only positive apoptotic cells were scattered leukocytes or Kupffer cells (arrow, F). Bars = 20 µm. Figures are representative of six animals per group carried out in three independent investigations. CV, central vein.

Figure 2

The effect of 24-h fasting of mice on the (A) processing of hepatic procaspase-3 (p32) to the active form (p17) by Western blot and (B) hepatic DNA laddering induced by APAP (530 mg/kg; 5 h). The effect of 24 h-fasting of mice on the serum level of (C) K18 apoptotic fragments (pmol/mL), (D) total HMGB1 content (ng/mL) and (E) ALT activity (U/L) after APAP treatment (530 mg/kg; 5 h). (F) The effect of APAP (530 mg/kg) on hepatic ATP content was determined over 0–5 h in fed (solid line) and fasted mice (dotted line). Figures are representative and data is given as mean ± SD of six mice per group carried out in three independent investigations. Statistical significance was assigned as *P < 0.05, **P< 0.01 relative to time-matched controls.

Table 1 Summary and comparison of APAP-induced effects in age-matched fasted and nonfasted male CD-1 mice and Z-VAD.fmk (10 mg/kg) pretreated fed mice (APAP 530 mg/kg; 5h and 24 h).^a

Glutathione Predepletion Does Not Inhibit the APAP-Induced Apoptotic Response in Fed Mice

Fasting of mice for 24 h resulted in significant decreases in both basal hepatic GSH and ATP levels (Table 1), and the consequences of this on APAP-induced apoptosis were explored. Hepatic GSH content was predepleted with DEM to fasting levels in fed mice. This had no effect on hepatic ATP (Figure 3C, D). In saline pretreated mice, procaspase-3 processing was first significant at 3 h (32.5% ± 7.8% active fragment of total) and observed 5 h after treatment (25.8% ± 4.5% active fragment of total). In DEM-pretreated mice, procaspase-3 processing was observed 2 h (26.1% ± 3.2% active fragment of total) and 3 h (23.6% ± 5.8% active fragment of total) after treatment (Figure 3A). There was no significant difference in procaspase-3 processing between mice predosed with saline or DEM. The earlier pattern of caspase activation was mirrored by hepatic DNA laddering (Figure 3B), serum K18 fragment abundance and total HMGB1 content (Figure 3E, F). There was no significant difference between K18 cleavage (Figure 3E), serum HMGB1 levels (Figure 3F) and ALT activity induced by APAP in saline (2370 ± 401 U/L) or DEM (3001 ± 763 U/L) pretreated mice at 5 h.

Figure 3

The effect of DEM predepletion (dotted line) of hepatic GSH on APAP-induced (530 mg/kg; 0–5 h) apoptotic and necrotic cell death compared with saline treatment (solid line) in fed male CD-1 mice. Representative (A) Western analysis of hepatic processing of procaspase-3 (p32) to the active form (p17) and (B) agarose gel analysis of hepatic DNA laddering. The effect of GSH predepletion on APAP-induced (C) hepatic GSH depletion, (D) hepatic ATP depletion and serum levels of (E) caspase-cleaved K18 fragments (pmol/mL) and (F) serum HMGB1 content (ng/mL) was determined simultaneously 0–5 h after APAP treatment. Data are given as mean ± SD of six mice per group carried out in three independent investigations. Statistical significance was assigned relative to vehicle treated controls (*P < 0.05, **P< 0.01 and ***P< 0.005) or between DEM and saline pretreated mice (†P < 0.05) at that time point.

Hepatic ATP Repletion in Fasted Mice Restores an APAP-Induced Apoptotic Response

The effect of basal hepatic ATP depletion resulting from fasting of mice undergoing APAP-induced hepatocyte apoptosis was investigated. When glucose and glycine were administered to fasted mice (glucose is a glycolytic substrate and glycine assists membrane stabilization), the basal level of hepatic ATP was significantly elevated from 17.1 ± 5.3 to 24.4 ± 3.7 nmol/mg. This resulted in processing of procaspase-3 (10.3% ± 5.4% active fragment of total) and hepatic DNA laddering, both absent in fasted mice treated with APAP alone (Figure 4A, B). Glucose/glycine administration had no inhibitory effect on APAP-induced GSH depletion (Figure 4C). K18 cleavage was significantly elevated from control in glucose/glycine-supplemented APAP-treated, fasted mice (Figure 4D). Hepatocyte necrosis (serum HMGB1 content and ALT activity) was also significantly decreased in fasted mice dosed with glucose/glycine with APAP (Figure 4E, F).

Figure 4

The effect of glucose and glycine administration (1000/100 mg/kg; 1 h pre-APAP) to fasted male CD-1 mice on APAP-induced (530 mg/kg; 5 h) apoptosis and necrosis. (A) Processing of hepatic procaspase-3, (B) hepatic DNA laddering, (C) hepatic GSH depletion (nmol/mg), (D) serum caspase-mediated K18 fragment level (pmol/mL), (E) total serum HMGB1 content (ng/mL) and (F) serum ALT activity (U/L). Figures are representative and data is given as mean ± SD of six mice per group carried out in three independent investigations. Statistical significance was assigned relative to vehicle-treated controls or between APAP alone and APAP + glucose/glycine (*P < 0.05, **P < 0.01 and ***P < 0.005).

Fasting Is Associated with the Inhibition of Caspase-Dependent HMGB1 Oxidation and the Recruitment of Inflammatory Cells after APAP Treatment

Caspase-dependent oxidation of the sulfhydryl group of C106 within the cytokine domain of HMGB1 (Figure 5A) is an important mechanism in neutralizing the immune-stimulatory potential of HMGB1 within apoptotic cells (27). We tested the hypothesis that induction of an inflammatory response after APAP-induced hepatocyte death is more intense in fasted compared with fed mice, based on a lack of oxidized HMGB1 in the fasted animals. Western blot of differential separation by nonreducing SDS-PAGE (27) and LC-MS/MS analysis of fasted mice dosed with APAP revealed that the only serum molecular form of HMGB1 present was the reduced form (Figure 5B). The predominant form of HMGB1 in the sera of fed mice dosed with APAP was the oxidized, immune-tolerant form (Figure 5B). Within the reduced HMGB1 protein, a peptide of 1947.3 Da was characterized by MS/MS containing C106. Additional MS/MS analysis identified peptides of 1963.3, 1979.3 and 1995.3 Da, present in only the oxidized and not the reduced form of HMGB1. The progressive increase in 16 amu corresponded to the addition of oxygen to C106 to produce the sulfonic, sulfenic and sulfinic acid—containing residues (Figure 5C, D).

Figure 5

Characterization of the cysteine 106 HMGB1 oxidation status with the sera of APAP (530 mg/kg; 5 h) dosed mice. (A) Schematic overview of the location of cysteine 106 within the cytokine domain of murine HMGB1, which is characterized by MS/MS. (B) Nonreducing SDS-PAGE separation and Western analysis of reduced and oxidized HMGB1 isolated by immunoprecipitation from the sera of fed and fasted APAP-dosed mice (530 mg/kg; 5 h) and characterized by LC-MS/MS. HMGB1 was also isolated from Z-VAD.fmk pretreated APAP-dosed mice. (C) MS/MS analysis of oxidized murine HMGB1 peptide 97–112 from a fed APAP-treated mouse containing the cysteine 106 sulfonic acid. (D) MS/MS analysis of reduced murine HMGB1 peptide 97–112 containing the cysteine 106 thiol from an APAP-treated mouse that was prefasted for 24 h. MS/MS and Western figures are representative of six mice per group carried out in three independent investigations.

The caspase dependency of C106 oxidation was confirmed with the use of a pan-caspase inhibitor Z-VAD.fmk in APAP-treated, fed mice. This treatment resulted in only the reduced molecular form of HMGB1 being detected within these mice (Figure 5B). In parallel with this result, a significant increase in serum levels of full-length K18 and ALT activity (necrosis biomarkers) was observed in mice when only the reduced form of HMGB1 was present (i.e., in fasted and caspase-inhibited fed mice). Histological examination confirmed extensive centrilobular cell loss with the presence of occasional necrotic cells in fasted mice (average score 2.5) and fed mice cotreated with Z-VAD.fmk (average score 2.2) at 24 h after treatment, whereas fed mice did not exhibit any centrilobular cell loss and/or necrotic cells at this stage (Table 1 and Figure 6A–C). Fed mice also did not show any evidence of inflammatory cell infiltration (Figure 6A), whereas fasted mice and fed mice cotreated with Z-VAD.fmk showed hepatic leukocyte recruitment, indicated by the presence of neutrophils rolling along the endothelial cell layer of central veins and portal veins (Figure 6B–D) and occasional emigrated centrilobular neu-trophils. Data in Table 1 support these findings, showing the high circulating levels of TNF-α and low levels of IL-6 in fasted APAP-treated versus Z-VAD.fmk and APAP-cotreated fed mice, as well as an increase in the serum levels of hyper-acetylated HMGB1 (an inflammatory biomarker) in contrast to the observed findings with fed mice (Table 1).

Figure 6

Histological determination of the effect of fasting and Z-VAD.fmk and anti-HMGB1 coapplication on APAP-induced hepatic changes in mice, 24 h after treatment. (A) In fed mice, there is no evidence of hepatocyte necrosis and the liver looks unaltered apart from an increased number of mitotic hepatocytes (arrow). There is no evidence of inflammatory cell recruitment into the liver. (B,C) Mice fasted for 24 h before treatment (B) and mice treated with APAP + Z-VAD.fmk show evidence of marked hepatocyte loss (score 3) and exhibit neutrophils in the lumen and rolling along endothelial cells (arrows) of central and portal veins, indicating leukocyte recruitment into the liver. (D) Mouse fasted for 24 h before APAP treatment. Closer view of a central vein with numerous neutrophils rolling along endothelial cells (arrows). (E). Treatment of fasted mice with APAP and anti-HMGB1 is associated with evidence of marked hepatocyte loss (score 3), but not of inflammatory cell recruitment into the liver. (F) After treatment of fasted mice with APAP and IgY; however, evidence of marked hepatocyte loss (score 3) and evidence of leukocyte recruitment, represented by neutrophils rolling along endothelial cells (arrows) of central and portal veins, is seen. Hematoxylin and eosin stain. Bars = 20 µm. PA, portal area; CV, central vein. Figures are representative of six animals per group carried out in three independent investigations.

Neutralizing Circulating HMGB1 Prevents Inflammatory Cell Recruitment after APAP Treatment

We investigated whether modulation of the level of the reduced form of HMGB1 had any effect on the presence and degree of hepatic inflammatory cell recruitment in fasted mice 24 h after treatment. There was significant reduction in the levels of serum hepatic necrosis markers (ALT activity and full-length K18) in the APAP + anti-HMGB1 antibody group compared with the APAP + control IgY group (Figure 7B, C). Treatment of animals with a neutralizing antibody to HMGB1 resulted in increased survival after APAP challenge compared with that observed in IgY-treated animals (Figure 7A). The histological findings were in accordance with these results. Although centrilobular cell loss was seen in the livers of both groups, this was more pronounced in the APAP + control IgY group (average score 2.7) compared with the APAP + anti-HMGB1 antibody group (average score 2.2) (Figure 6E, F). In both APAP-treated groups significant elevation in the serum level of TNF-α was observed (Figure 7E); however, the level of TNF-α was greater in the control group receiving APAP + IgY (232.3 ± 54.2 pg/mL) compared with the APAP + anti-HMGB1 group (121.0 ± 16.7 pg/mL). Mice treated with APAP + anti-HMGB1 also showed significant elevations in the serum level of IL-6 (246.2 ± 36.8 pg/mL) (Figure 7F). These data in conjunction with data from serum indicate a systemic inflammatory response, which was confirmed by the histological findings. Whereas the APAP + control IgY group exhibited moderate numbers of rolling neutrophils in central and portal veins, there was no evidence of inflammatory cell recruitment in the APAP + anti- HMGB1 group (Figure 6E, F), a finding similar to that observed in fed mice.

Figure 7

Effect of HMGB1 neutralization on APAP (530 mg/kg; 24 h) hepatotoxicity. Mice were treated with an HMGB1-neutralizing antibody or a control IgY antibody 2 h after APAP treatment and (A) percent (%) survival over 24 h (Fed mice + APAP solid line; fasted mice + APAP/IgY, large dashed line; fasted mice + APAP/anti-HMGB1, small dashed line), (B) serum full-length K18 level (pmol/mL), (C) serum ALT activity (U/L), (D) serum total HMGB1 content, (E) serum TNF-α level (pg/mL) and (F) serum IL-6 level (pg/mL) were recorded simultaneously within the same group of mice. Data are representative of mean ± SD of six mice per group carried out in three independent investigations. Statistical significance was assigned between groups as appropriate. *P < 0.05, **P < 0.01 and ***P < 0.005.

Discussion

The reaction of the liver to an insult, disease process or experimental conditions often involves mixed modes of cell death (13). We have investigated whether fasting of mice for 24 hours can inhibit APAP-induced caspase activation and apoptosis through the depletion of basal ATP (11,12,28) and promote the induction of the infiltration of inflammatory cells. The data presented provide a rational biochemical explanation why apoptosis, necrosis and inflammation are seen to differing degrees in either fasted or fed models of APAP-induced hepato-toxicity. Along with other susceptibility-factors such as hepatic GSH and CYP450 content, the outcome of these investigations supports the clinical observation that food deprivation may alter susceptibility to APAP overdose (33).

Fasting of animals before toxicological investigation has several advantages, particularly reduced data variability. However, nonclinical safety evaluation routinely occurs in healthy animals, with controlled health status and diet, at doses that are higher than those targeted for human use. With respect to the investigation of DILI through reactive metabolite formation, fasting of animals can profoundly affect the toxicological response to the drug by decreasing hepatic GSH (34) and ATP (35), altering CYP450 expression (36) and the downregulating gene expression associated with apoptosis (37). In the present study we did not observe a significant difference between ALT levels in fasted and fed mice. However, in fasted mice the interanimal ALT variation was decreased relative to that of fed mice. Greater HMGB1 and full-length K18 release were measured in the fasted compared with fed APAP-treated mice, suggesting ALT activity is not as sensitive an indicator of liver necrosis.

We have recently shown APAP-induced hepatocyte apoptosis in a fed CD-1 mouse model of hepatotoxicity with no histological evidence for inflammatory cell recruitment (6), despite the release of DAMPs, such as HMGB1, into the circulation. Rather, evidence of liver regeneration was observed. In our previous study using fed mice, we found evidence of a potential protective role for apoptosis in APAP-induced hepatotoxicity, based on the occurrence of apoptotic hepatocyte death at the early stage (3 hours) after treatment. Considering that hepatocyte apoptosis including phagocytosis does not on average take longer than 3 hours (38); these findings suggest that apoptosis is an early effect of APAP on hepatocytes. It therefore cannot be ruled out that we missed the presence of apoptotic hepatocytes at a very early stage (less than 3 hours) after treatment. If apoptosis had occurred between the 5- to 24–hour time points investigated in fasted mice and the morphological evidence of apoptosis had been missed (39), K18 cleavage should have been detected at the 24-hour time point, based on the demonstrated long half-life of fragmented K18. Apoptotic hepatocytes, as apoptotic bodies, are rapidly phagocy-tosed by both hepatocytes and Kupffer cells, but could also be phagocytosed by circulating monocytes, in particular in the liver as an organ with extensive constant blood throughput (38). The intense blood supply is also the likely cause for the very low number of necrotic cells observed histologically, because the cell debris will be removed from the liver by the blood.

In the present study, the use of a pan-caspase inhibitor led to enhanced necrosis in fed animals (in which apoptosis is normally observed). In studies of TNF-α and Fas-mediated fulminant hepatic failure, inhibition of caspases or overexpression of the antiapoptotic protein Bcl-2, leads to complete elimination of apoptosis and injury reduction (40,41). However, the apoptotic pathway initiated by TNF or FasL is via death receptors and generally results in slower depletion of ATP levels. The hepatic damage induced by APAP, mediated through mitochondrial permeability transition pore opening, is accompanied by both caspase-dependent apoptotic signaling and oncotic necrosis, and the two modes of cell death are frequently observed in many pathophysiological settings (41). The rapid depletion of ATP switches the predominant mode of cell death toward oncotic necrosis, which is well recognized (42). Also, ATP supplementation in vitro can revert FasL-induced necrosis back to apoptosis as the dominant form of cell death (43).

It has been proposed that the most prominent biochemical consequence of fasting is a near complete depletion of hepatic glycogen, used to generate ATP via glycolysis and the citric acid cycle (44). Hepatocellular glycogen depletion in fasted mice was confirmed histologically in the present study, coupled with a significantly decreased basal hepatic ATP. We also observed a significant decrease in basal hepatic GSH in these animals. The level of APAP covalent binding was not significantly different in fasted and fed mice. As seen in vitro, the basal level of hepatic ATP was increased by predosing APAP-treated fasted mice with glucose and glycine (45), and apoptosis was observed in these animals. Moreover, the levels of serum markers used to quantify necrosis also decreased after glucose/glycine coadministration. It is important that even with the increase in basal ATP content, the major form of cell death induced by APAP was necrosis. Supplementation of glucose/glycine to fasted mice could not restore the quantitative level of apoptosis to that observed in fed mice treated with APAP alone. The data resulting from this investigation suggest that depletion of hepatic ATP to below a significant threshold level by fasting prevents the induction of an apoptotic response. Moreover, our data suggest that only small increases in hepatic ATP can drive the switch from necrosis to an apoptotic pathway with glycolytic substrates. This result is consistent with other reported data showing that boosting the hepatic ATP content by 15–20% of control levels prevents necrotic cell death during hepatic ischemia-reperfusion injury (46).

The effect of hepatic GSH depletion before APAP treatment, without altering ATP levels, was investigated. Previous depletion of GSH in fed mice to a similar level observed after fasting did not result in inhibition of APAP-induced apoptosis. The time of apoptosis onset was observed at 2 hours compared with 3 hours in saline-pretreated mice. The presence of the observed caspase cleavage and DNA laddering was lost as apoptosis progressed to necrosis in the same manner as we have observed at late time points in our previously reported investigation (6). Furthermore, the sensitization of hepatocytes to apoptosis via TNF-α by GSH depletion has previously been shown (47).

The immunostimulatory properties of HMGB1 have been found to vary depending on the oxidation status of the sulphydryl group on C106. Recent reports also highlight the critical presence of C106 for the proinflammatory function of HMGB1 through TLR4 signaling (26). Oxidation of the sulphydryl group is an intracellular posttranslational modification and has been shown to be a caspase-directed process to promote immune tolerance in vitro (27). We have demonstrated by Western blot and LC-MS/MS that the oxidized form of HMGB1 is the predominant form in the blood of fed mice dosed with APAP, and oxidation depends on caspase activation, an observation that has previously been made only in vitro (27).

Infiltration of neutrophils into the liver, without distinct evidence of hepatic regeneration, was seen in fasted but not fed mice after APAP treatment. Serum TNF-α concentrations were higher in fasted mice treated with APAP. Other studies have demonstrated that IL-6 plays a protective role in APAP hepatotoxicity, whereas TNF-α is detrimental (17,19). Significantly elevated serum levels of IL-6 and hepatic regeneration were seen in APAP-treated, fed mice. The data presented implicate an important role for the oxidation status/inflammatory potential of circulating HMGB1 in the cytokine expression profile. Caspase-inhibited mice with circulating reduced C106-HMGB1 demonstrated the same hepatic inflammatory cell infiltration at 24 hours after APAP treatment as did fasted mice and the same pattern of IL-6 and TNF-α in their sera. Furthermore, the serum level of acetylated-HMGB1 (immune cell derived) supported this observation and was upregulated in fasted compared with fed mice treated with APAP.

Fed mice did not display inflammatory cell recruitment into the liver after APAP hepatotoxicity and showed APAP-in-duced apoptotic hepatocyte death. However, when treated with a caspase inhibitor the fed mice displayed no evidence of oxidized C106 HMGB1 and demonstrated the same hepatic inflammatory cell recruitment at 24 hours after APAP as fasted mice. Fed mice were also found to display the same levels of IL-6 and TNF-α in their sera. The antibody-mediated neutralization of the inflammatory potential of HMGB1 in a murine model of endotoxin lethality and protection has previously been reported (48). Likewise, we found that antibody-mediated neutralization of HMGB1 in fasted mice modulated the inflammatory response associated with APAP hepatotoxicity. These findings are in accordance with data reported in models of APAP hepatotoxicity by others (22,48). However, the observed increase in IL-6 after anti-HMGB1 treatment with APAP requires additional investigation. One possibility is that HMGB1 sequesters the hepatic regenerative effects of IL-6 by direct protein-protein binding. The binding of HMGB1 and alteration of cytokine function has previously been described (49). It is also important to note that when HMGB1 was identified as the acetylated derivative (immune-cell derived [6]), C106 was also in the reduced thiol status. These findings confirm a significant role played by reduced HMGB1 in the induction of inflammatory cell recruitment and highlight important mechanisms by which this response is perturbed by HMGB1 oxidation during apoptosis, which modulates the overall extent of DILI (Figure 8). In particular, given the important observation that C106 plays a critical role for TLR4 binding and that TLR4^−/− mice are resistant to APAP toxicity (50).

Figure 8

Schematic overview summarizing the findings within this investigation. APAP-in-duced hepatic apoptosis and necrosis in the fed CD-1 mouse model, which can be quantified by the serum biomarker K18. This is accompanied by the caspase-dependent oxidation of HMGB1 (HMGB1-SO₃H), which prevents an inflammatory response. The fasting of mice before APAP induces a depletion in basal hepatic ATP levels, which does not permit apoptosis activation and HMGB1 oxidation. The resulting reduced HMGB1 (HMGB1-SH) activates inflammatory cells and promotes the hepatic inflammatory response after hepatocyte death, leading to exacerbated injury.

The modeling and prediction of human variability in DILI in animal models remains a major goal for safe drug development. The biological responses in fasted or fed animals/humans may have different mechanistic relevance both clinically and preclinically, a situation that is important in the consideration of adverse reactions in which host-specific factors or disease play a dominant role in the pathogenesis of DILI (51). Fed and fasted preclinical models are important and equally valid; however, more mechanistic information is required for both. To understand the signals by which a controlled apoptotic/regenerative response to chemical insult can be overwhelmed by a necrotic response may allow the development of novel intervention therapies for cases of APAP overdose and help our general understanding of chemically induced liver injury.

Disclosure

The authors declare that they have no competing interests as defined by Molecular Medicine, or other interests that might be perceived to influence the results and discussion reported in this paper.