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Abstract

  1. Top of page
  2. Abstract
  3. Methods
  4. Results
  5. Discussion
  6. References
  7. Appendix

Obstructive sleep apnoea (OSA) leads to chronic intermittent hypoxia (CIH) during sleep. Obstructive sleep apnoea has been associated with liver injury. Acetaminophen (APAP; known as paracetamol outside the USA) is one of the most commonly used drugs which has known hepatotoxicity. The goal of the present study was to examine whether CIH increases liver injury, hepatic oxidative stress and inflammation induced by chronic APAP treatment. Adult C57BL/6J mice were exposed to CIH or intermittent air (IA) for 4 weeks. Mice in both groups were treated with intraperitoneal injections of either APAP (200 mg kg−1) or normal saline daily. A combination of CIH and APAP caused liver injury, with marked increases in serum alanine aminotransferase, aspartate aminotransferase (AST), γ-glutamyl transferase and total bilirubin levels, whereas CIH alone induced only elevation in serum AST levels. Acetaminophen alone did not affect serum levels of liver enzymes. Histopathology revealed hepatic necrosis and increased apoptosis in mice exposed to CIH and APAP, whereas the liver remained intact in all other groups. Mice exposed to CIH and APAP exhibited decreased hepatic glutathione in conjunction with a fivefold increase in nitrotyrosine levels, suggesting formation of toxic peroxynitrite in hepatocytes. Acetaminophen or CIH alone had no effect on either glutathione or nitrotyrosine. A combination of CIH and APAP caused marked increases in pro-inflammatory chemokines, monocyte chemoattractant protein-1 and macrophage inflammatory protein-2, which were not observed in mice exposed to CIH or APAP alone. We conclude that CIH and chronic APAP treatment lead to synergistic liver injury, which may have clinical implications for patients with OSA.

Obstructive sleep apnoea (OSA) is characterized by recurrent closure of the upper airway during sleep, leading to chronic intermittent hypoxia (CIH) and sleep fragmentation (Gastaut et al. 1966). Obstructive sleep apnoea is present in 2–9% of women and 4–24% of men in the USA, but the prevalence may exceed 50% in the obese population (Bixler et al. 2001; Punjabi et al. 2002; Young et al. 1993). Obstructive sleep apnoea has been independently associated with development of hypertension, cardiovascular disease, stroke and diabetes mellitus type 2 (Nieto et al. 2000; Peppard et al. 2000; Shahar et al. 2001; Punjabi et al. 2003, 2004; Punjabi & Polotsky, 2005; Marin et al. 2005; Yaggi et al. 2005). There is also emerging evidence that OSA is associated with chronic liver injury and non-alcoholic steatohepatitis (Singh et al. 2005; Tanne et al. 2005; Tatsumi & Saibara, 2005; Jouet et al. 2007; Kallwitz et al. 2007; Zamora-Valdes & Mendez-Sanchez, 2007; Kheirandish-Gozal et al. 2008; Norman et al. 2008).

The CIH of OSA induces repetitive cycles of hypoxia and reoxygenation, leading to generation of reactive oxygen species (ROS) and systemic oxidative stress (Row et al. 2002; Lavie, 2003, 2004; Chen et al. 2005; Zhan et al. 2005; Prabhakar et al. 2007). We have previously developed a murine model of CIH (Polotsky et al. 2006) and have shown that CIH leads to both mild liver injury and elevations in biomarkers of oxidative stress (Li et al. 2007; Savransky et al. 2007b,c; Jun et al. 2008). We have also shown that CIH may act as a ‘second hit’, amplifying the liver injury induced by a single high dose of acetaminophen (APAP; known as paracetamol outside the USA; Savransky et al. 2007c). However, the effects of CIH on acute APAP overdose are of limited clinical significance given that APAP overdose is relatively rare. In contrast, APAP is among the most commonly used pain-relievers, and an association between OSA and chronic intake of APAP can be frequently encountered.

Acetaminophen is metabolized in the liver. About 90% of a therapeutic dose of APAP is conjugated with glucuronic acid or sulphate and excreted into bile or blood (Jaeschke, 2005). The remaining 10% of APAP is metabolized by the hepatic cytochrome P450 system to N-acetyl-p-benzoquinone imine, which reacts with reduced glutathione (GSH) to form a GSH-adduct (Chen et al. 2003a; Jaeschke & Bajt, 2006). When APAP overdose occurs, N-acetyl-p-benzoquinone imine depletes mitochondrial GSH stores and leads to oxidative stress with predominant generation of peroxynitrite (Jaeschke & Bajt, 2006). Peroxynitrite is formed in the mitochondria from nitric oxide (NO) and superoxide and can be assessed by 3-nitrotyrosine (3-NT) levels. Inducible NO synthase (iNOS) may play a role in APAP liver toxicity (Michael et al. 2001; Jaeschke & Bajt, 2006). It is conceivable that CIH and APAP have a synergistic injurious effect on hepatocytes, causing severe oxidative stress with resultant tissue injury and inflammation.

The goal of the present study was to explore whether CIH exacerbates liver toxicity induced by chronic administration of APAP. We exposed C56BL/6J mice to CIH and APAP and examined: (1) serum activity of liver enzymes; (2) liver histopathology; (3) indices of oxidative stress, including GSH, 3-NT and lipid peroxidation; and (4) liver levels of pro-inflammatory cytokines.

Methods

  1. Top of page
  2. Abstract
  3. Methods
  4. Results
  5. Discussion
  6. References
  7. Appendix

Animals

A total of 38 wild-type, 6- to 8-week-old male, lean C57BL/6J mice purchased from Jackson Laboratory (Bar Harbor, ME, USA) were used in the study. The study was approved by the Johns Hopkins University Animal Care and Use Committee and complied with the American Physiological Society Guidelines for Animal Studies. For all blood samples, injections and surgical procedures, anaesthesia was induced and maintained with 1–2% isoflurane in 40% O2, administered through a facemask.

Experimental design

A gas control delivery system was designed to regulate the flow of room air, nitrogen and oxygen into customized cages housing the mice as previously described (Polotsky et al. 2003). A maximum of three mice were housed continuously in a single customized cage (dimensions 27 cm × 17 cm × 17 cm) with constant access to food and water. During each period of intermittent hypoxia (IH), the fractional inhaled O2 was reduced from 20.9 to 4.9 ± 0.1% over a 30 s period and then rapidly reoxygenated to room air levels in the subsequent 30 s period, resulting in 60 hypoxic events per hour. We have previously determined that an average arterial partial pressure of O2 over a hypoxic cycle was 51.7 ± 4.2 mmHg (Savransky et al. 2007a). Thus, our murine model mimicked CIH of severe OSA.

Food intake and body weight were monitored daily for each animal. All animals were kept in a controlled environment (22–24°C with a 12 h–12 h light–dark cycle; lights on at 09.00 h) on a standard Purina chow diet with free access to food and water. Eighteen mice were placed in the IH chamber for four consecutive weeks. Twenty mice were exposed to intermittent room air (IA, control groups) for 4 weeks in identical chambers (Table 1). IA was delivered at the same rate as hypoxic gases and produced an average arterial partial pressure of O2 of 99.7+/−2.1 mmHg (Savransky et al. 2007a). The CIH and IA states were induced during the light phase, alternating with 12 h of constant room air during the dark phase. During the last 2 weeks of exposure, nine mice exposed to CIH and ten mice exposed to IA were treated with daily intraperitoneal injections of APAP (Sigma-Aldrich Corp., St Louis, MO, USA) at 200 mg kg−1 in 0.3 ml of 0.9% sodium chloride, while nine mice exposed to CIH and ten mice exposed to IA were treated with daily intraperitoneal injections of placebo (0.3 ml of 0.9% sodium chloride). The dose was selected based on our pilot data demonstrating that APAP in a dose up to 300 mg kg−1 did not induce significant elevation of liver enzymes in male C57BL/6J mice (not shown).

Table 1.  Basic characteristics of C57BL/6J mice exposed to chronic intermittent hypoxia and acetaminophen
 PlaceboAcetaminophen
Intermittent airIntermittent hypoxiaIntermittent airIntermittent hypoxia
  1. *P < 0.05 between day 0 and day 14 or between day 0 and day 28 or between day 14 and day 28; **P < 0.01 between day 0 and day 14 or between day 14 and day 28; ***P < 0.001 between day 14 and day 28; †P < 0.001 between day 0 and day 28; ‡P < 0.05 and ‡‡P < 0.001 between intermittent air and intermittent hypoxia; and §P < 0.05 and §§P < 0.001 between acetaminophen and placebo.

n109109
Starting age (weeks)8888
Body weight (g)
  Day 0 24.2± 0.6 24.6 ± 0.6  24.7 ± 0.523.9 ± 1.2
  Day 14 26.4 ± 0.6* 22.5 ± 0.5*‡   26.7 ± 0.3 **  21.1 ± 0.5*‡
  Day 28 26.8 ± 0.7* 25.4 ± 0.8**25.8 ± 0.6      18.9 ± 0.5**†‡‡§§
Food intake (g day−1)
  Day 14 3.0 ± 0.1  2.2 ± 0.03‡‡ 3.1 ± 0.2    2.1 ± 0.01‡‡ 
  Day 28 2.9 ± 0.1 2.7 ± 0.1        2.4 ± 0.03***§§        1.4 ± 0.05***‡‡§§
Liver weight (g) 1.27 ± 0.05 1.06 ± 0.05‡‡ 1.14 ± 0.04    0.89 ± 0.03‡‡§
Liverweight/body weight (%) 4.72 ± 0.17 4.15 ± 0.1‡   4.43 ± 0.0.9    4.74 ± 0.16§ 
Serum total protein (g dl−1) 4.38 ± 0.06 4.99 ± 0.19‡ 4.84 ± 0.13 4.91 ± 0.11
Serum albumin (g dl−1) 3.04 ± 0.07 3.41 ± 0.11  3.33 ± 0.09 3.63 ± 0.16
Fasting blood glucose (mg dl−1) 228 ± 18 233 ± 25  194 ± 12  141 ± 12‡§

Sample collection

Mice were fasted for 5 h prior to terminal anaesthesia and blood sampling. Arterial blood (1 ml) was obtained by direct cardiac puncture under 1–2% isoflurane anaesthesia. Serum was separated and frozen at −80°C. After blood withdrawal, the animals were killed with pentobarbitone (60 mg, intraperitoneally). The liver was surgically removed and weighed. Liver tissue was either frozen in liquid nitrogen and kept at −80°C for further biochemical studies or fixed in buffered 10% formalin for histological examination.

Biochemical assays

Serum alanine aminotransferase (ALT), aspartate aminotransferase (AST) and γ-glutamyl transferase (GGT) activities, serum total and direct bilirubin, total protein and albumin levels were measured by the Clinical Chemistry Laboratory of the Johns Hopkins Bayview Medical Center. Fasting blood glucose was measured with Accu-Chek® Comfort Curve TM kit from Roche Diagnostics, Inc. (Indianapolis, IN, USA). The liver was homogenized using Omni EZ Connect Homogenizer (Omni International, Warrington, VA, USA). γ-Glutamyl transferase, 3-NT and malondialdehyde (MDA) levels were measured in total liver lysate with kits from Oxis Research (Portland, OR, USA). Tumour necrosis factor-α (TNF-α), monocyte chemoattractant protein-1 (MCP-1) and macrophage-inflammatory protein-2 (MIP-2) levels in total liver lysate were determined with ELISA kits from R&D Systems, Inc. (Minneapolis, MN, USA). Interleukin-6 (IL-6) levels in the liver were measured with an ELISA kit from RayBiotech, Inc. (Norcross, GA, USA).

Histology

Paraffin-embedded tissue was sectioned at 3–5 μm thickness and stained with Haematoxylin and Eosin (H&E). Semi-quantitative analysis of the H&E sections was performed by an experienced hepatopathologist (M.S.T.) without knowledge of treatment conditions or laboratory findings. The scoring system developed and validated for steatohepatitis in humans has been adapted for the study with minor modifications (Kleiner et al. 2005). The following scoring criteria have been used: steatosis (0, none; 1, 1–30% hepatocytes; 2, 31–60% hepatocytes; and 3, >60% hepatocytes), hepatocyte swelling (same as steatosis), lobular inflammation (0, none; 1, average of 1 inflammatory infiltrate; 2, average of 2–4 inflammatory infiltrates; 3, 5–10 inflammatory infiltrates; and 4, >10 inflammatory infiltrates per × 10 field) and necrosis (1, occasional necrotic cell; 2, numerous necrotic cells in zone 3; 3, confluent zone 3 necrosis; and 4, necrosis extending beyond zone 3). Terminal deoxynucleotidyl transferase biotin–dUTP nick end labelling (TUNEL) was performed in liver sections to detect apoptosis using the Fluorescein FragEL DNA Fragmentation Detection Kit (catalogue no. QIA39) from Calbiochem (San Diego, CA, USA). To determine the percentage of apoptotic cells, all cells on one section (10–50 mm2) were examined, and TUNEL-positive cells were counted under fluorescence microscopy at an excitation wavelength of 330–380 nm. Results are expressed as a number of TUNEL-positive cells/total cells × 100%.

Gene expression analysis

Total RNA was extracted from liver using TRIzol (Invitrogen, Carlsbad, CA, USA) and complementary DNA was synthesized using Advantage RT for PCR kit from Clontech (Palo Alto, CA, USA). Real-time reverse transcriptase-polymerase chain reaction PCR (RT-PCR) was performed with primers and probe specific for iNOS (Genbank accesssion number Mm00440488_m1), endothelial NOS (eNOS, Genbank accesssion number Mm0113492_g1) and 18S (Li et al. 2005a,b) from Applied Biosystems (Foster City, CA, USA). Of note, 18S is an optimal housekeeping gene for liver injury (Pelletier et al. 2000). The mRNA expression levels were normalized to 18S ribosomal RNA (rRNA) concentrations using the following formula:

  • image

where Ct is the cycle threshold.

Statistical analyses

All values are reported as means ±s.e.m. Statistical comparisons between four groups of mice (CIH-APAP, IA-APAP, CIH-Placebo and IA-Placebo) were performed by a general linear model ANOVA across two independent variables, hypoxia and APAP, followed by Tukey's post hoc test. Comparisons between day 0, day 14 and day 28 of mouse exposure were performed using repeated-measures ANOVA. A P value of less than 0.05 was considered significant.

Results

  1. Top of page
  2. Abstract
  3. Methods
  4. Results
  5. Discussion
  6. References
  7. Appendix

Mice exposed to CIH exhibited a decrease in food intake and 9–11% weight loss during the first 2 weeks of the experiment, whereas IA control mice gained 8–9% of the starting body weight (Table 1). During the last 2 weeks of the experiment, mice receiving placebo showed adaptation to the CIH stimulus, with restored food intake and weight gain (Table 1). Administration of APAP from day 14 to 28 resulted in a mild reduction in food intake in mice exposed to IA, while body weight did not differ significantly from the placebo group. In contrast, mice exposed to CIH and APAP showed a dramatic decrease in food intake and body weight by day 28 (Table 1). Chronic intermittent hypoxia led to a decrease in liver weight in both placebo and APAP-injected mice.

In placebo-treated mice, CIH doubled serum AST levels and slightly increased serum total protein levels, whereas serum ALT, GGT, total bilirubin, albumin and fasting blood glucose levels were not altered (Fig. 1 and Table 1). Mice treated with APAP and exposed to IA exhibited normal levels of liver enzymes and a mild increase in serum total bilirubin level (Fig. 1). Chronic intermittent hypoxia significantly increased liver injury induced by APAP, as evidenced by a threefold increase in serum ALT, a decline in fasting blood glucose, a marked increase in serum total bilirubin and appearance of direct bilirubin (0.06 ± 0.02 mg dl−1), which was absent in all other animal groups (Fig. 1 and Table 1).

image

Figure 1. The effects of chronic intermittent hypoxia (CIH) and acetaminophen (APAP) on serum enzymatic activity of alanine aminotransferase (ALT; A), aspartate aminotransferase (AST; B), γ-glutamyl transferase (GGT; C) and serum levels of total bilirubin (D) *P < 0.05 and **P < 0.001 between CIH and intermittent air control; ‡P < 0.001 between APAP and placebo.

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Histological examination of the livers revealed normal hepatic architecture, with no significant lobular or portal inflammation, steatosis or necrosis and mild hepatocyte swelling in mice exposed to IA and placebo (Fig. 2A), IA and APAP (Fig. 2B) and CIH and placebo (Fig. 2C). In contrast, animals exposed to CIH and APAP showed striking liver injury, with discernable swelling of hepatocytes (average score of 3 ± 0 versus 1.3 ± 0.2 in all other groups, P < 0.001) and necrosis (average score of 1.5 ± 0.6 versus 0 in other groups of animals, P= 0.01). The extent of liver necrosis varied from occasional hepatocytes to large confluent areas in zone 3 around the central veins (Fig. 2D). Combined CIH-APAP injury led to a twofold increase in apoptosis in the liver, whereas CIH or APAP alone had no effect (Fig. 3).

image

Figure 2. Liver histology in mice exposed to intermittent air (IA) and placebo (A), IA and APAP (B), CIH and placebo (C) or CIH and APAP (D) The livers of mice exposed to APAP showed marked hepatocellular necrosis in zone 3 only if they were also subjected to CIH (D). Necrosis was not seen after exposure to IA with or without APAP (A and B) or to CIH without APAP (C). All images H&E; scale bar represents.

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image

Figure 3. Terminal deoxynucleotidyl transferase biotin–dUTP nick end labelling (TUNEL) in liver tissue of mice exposed to intermittent air and placebo (A) or CIH and APAP (B). White arrows point to apoptotic cells. Scale bar represents. C, quantification of the effects of CIH and APAP on the percentage of apoptotic liver cells. *P < 0.01 between CIH plus APAP and all other groups.

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Liver tissue was assessed for markers of oxidative stress and inflammation. In mice injected with placebo, CIH did not have a significant effect on GSH and 3-NT levels, although there was a trend towards increased MDA (P= 0.09, Fig. 4). In mice exposed to IA and APAP, there was no evidence of oxidative stress in the liver. Simultaneous exposure to CIH and APAP depleted GSH stores by 26% and induced a fivefold increase in 3-NT levels in the liver, compared with APAP alone, while MDA levels were not affected (Fig. 4). An increase in 3-NT could be related to up-regulation of NO biosynthesis; therefore, NOS expression was examined. Inducible NOS mRNA was undetectable, whereas eNOS mRNA was expressed at identical levels (eNOS/18S mRNA ratio = 0.00017 ± 0.00004) in all groups of mice. Taken together, these results suggest that CIH significantly exacerbates oxidative stress induced by APAP, preferentially affecting reactive nitrogen species.

image

Figure 4. The effects of CIH and APAP on levels of reduced glutathione (GSH; A), 3-nitrotyrosine (3-NT; B) and malondialdehyde (MDA; C) in the liver *P < 0.05 between CIH and intermittent air control; †P < 0.01 between CIH and intermittent air control in mice treated with APAP and between placebo and APAP in mice subjected to CIH.

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Chronic intermittent hypoxia alone did not induce noticeable inflammatory changes in liver parenchyma. Control IA animals exposed to APAP showed increases in liver IL-6 and TNF-α levels, whereas levels of chemokines MCP-1 and MIP-2 were similar to those of mice treated with placebo (Fig. 5). Animals exposed to both CIH and APAP exhibited a sixfold increase in hepatic MCP-1 and a nearly 40% increase in hepatic MIP-2 (Fig. 5). Levels of TNF-α and IL-6 were similar in hypoxic and normoxic mice treated with APAP.

image

Figure 5. The effects of CIH and APAP on levels of tumour necrosis factor-α (TNF-α; A), interleukin-6 (IL-6; B), monocyte chemoattractant protein-1 (MCP-1; C) and macrophage inflammatory protein-2 (MIP-2; D) in the liver *P < 0.05 between CIH and intermittent air control.

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Discussion

  1. Top of page
  2. Abstract
  3. Methods
  4. Results
  5. Discussion
  6. References
  7. Appendix

We have previously shown that CIH leads to mild liver injury in C57BL/6J mice and that CIH can greatly exacerbate toxicity of APAP administered as a single high dose (600 mg kg−1). The purpose of this study was to determine whether a combination of CIH and chronic treatment with APAP can cause liver dysfunction and to characterize mechanisms involved. The study resulted in several novel findings. First, mice exposed to CIH and chronic APAP developed liver injury, with elevation of serum ALT and histopathological evidence of increased apoptosis and hepatocellular necrosis, which was not observed in mice exposed to CIH alone or APAP alone. Second, mice exposed to CIH and treated with APAP suffered oxidative injury to the liver, with accumulation of 3-NT and decreased GSH, which was not observed in the other groups of mice. Third, mice exposed to CIH and treated with APAP exhibited increased levels of chemokines MCP-1 and MIP-2 in the liver, compared with the other groups of mice. In the discussion below, we will examine the possible mechanisms of CIH interaction with APAP and discuss clinical implications of our work.

Chronic intermittent hypoxia, APAP and oxidative stress

Obstructive sleep apnoea is associated with increased serum levels of malondialdehyde (MDA) and 8-isoprostanes (Lavie, 2003; Lavie et al. 2004). Experimentally induced CIH in rodents causes lipid peroxidation in the brain, carotid bodies, adrenal glands and myocardium (Row et al. 2002; Zhan et al. 2005; Chen et al. 2005; Kumar et al. 2006; Peng et al. 2006a,b). We have shown that CIH leads to increased lipid peroxidation in the liver (Li et al. 2007; Jun et al. 2008) and that liver injury in CIH is associated with increased levels of lipid peroxidation products (Savransky et al. 2007c). Repetitive cycles of hypoxia and reoxygenation increase ROS generation by the mitochondrial electron transport chain, which is the main source of ROS during normal metabolism (Chandel & Schumacker, 2000; Chen et al. 2003b; Lavie, 2003; Prabhakar & Kumar, 2004; Lahiri et al. 2006). Microsomal enzyme systems, such as CYP2E1, CYP4A, xanthine oxidase, nitric oxide synthases and NADPH oxidase, can also participate in the formation of ROS in the liver (Robertson et al. 2001; Lavie, 2003; Peng et al. 2006a; Choi et al. 2006; Jun et al. 2008). Our present data suggest that, although CIH alone tends to increase lipid peroxidation, APAP alone did not have a similar effect. Moreover, APAP abolished a CIH-induced increase in lipid peroxidation. We speculate that this unexpected finding is a result of perturbations in superoxide metabolism. Indeed, superoxide has to be converted to hydrogen peroxide and hydroxyl radicals to induce lipid peroxidation (Halliwell & Gutteridge, 2007). Several reports indicate that hydrogen peroxide and hydroxyl radicals, as well as lipid peroxidation, are of limited relevance in APAP toxicity, because superoxide is rapidly utilized in the peroxynitrite reaction: NO+ O2•−[RIGHTWARDS ARROW] ONOO, which is several times faster than the superoxide dismutase (SOD) reaction (Jaeschke & Bajt, 2006; Halliwell & Gutteridge, 2007). Of note, conflicting reports indicate that SOD1 is necessary for APAP-induced liver injury (Lei et al. 2006). Nevertheless, it is conceivable that APAP shunts CIH-induced ROS away from lipid peroxidation to the mitochondrial peroxynitrite reaction.

Peroxynitrite leads to rapid protonation, depletion of antioxidant defense, and nitration and oxidation of amino acids (Beckman & Koppenol, 1996; Alvarez & Radi, 2003; Halliwell & Gutteridge, 2007). Nitration of tyrosine, resulting in 3-NT, is considered a reliable biomarker of peroxynitrite generation. Peroxynitrite and 3-NT damage the mitochondria and cytoskeleton, leading to liver injury (Greenacre & Ischiropoulos, 2001; Cover et al. 2005; Halliwell & Gutteridge, 2007). Exposure of isolated and perfused rat liver to hypoxia resulted in 3-NT accumulation in pericentral regions (Arteel et al. 1999), but the effects of CIH and OSA on hepatic 3-NT levels were not previously studied. In contrast, the role of 3-NT in APAP toxicity is well known (Hinson et al. 1998; Knight et al. 2001; Cover et al. 2005; Jaeschke & Bajt, 2006). We have shown that: (1) CIH alone induces mild liver injury without an increase in 3-NT; (2) chronic administration of APAP does not induce liver injury or increases 3-NT; and (3) a combination of CIH and APAP leads to overwhelming liver injury with necrosis in association with a four- to fivefold increase in 3-NT. Since peroxynitrite is generated predominantly in mitochondria (Cover et al. 2005), our findings suggest that CIH and APAP interact to induce mitochondrial dysfunction, promoting liver injury. One possibility is that CIH inhibits APAP metabolism, resulting in higher APAP levels, which would be a simple and elegant explanation of the CIH–APAP synergy. However, APAP levels have not been measured, which is a limitation of the study.

What are the mechanisms of an increase in hepatic peroxynitrite levels during CIH and APAP treatment? One mechanism would be accelerated generation of peroxynitrite owing to an increase in NO production. There is no consensus in the literature on the effects of CIH on NOS. In rodents, CIH increases levels of eNOS in pulmonary vasculature (Snow et al. 2008), decreases eNOS expression in penile tissue (Soukhova-O’Hare et al. 2008) and decreases neuronal NOS expression in the hippocampus (Tjong et al. 2008). Patients with OSA exhibit decreased levels of eNOS and increased levels of iNOS in venous endothelial cells, and these changes are reversible by CPAP treatment (Jelic et al. 2008). The effect of CIH on NOS isoforms in the liver has not been previously assessed. Acetaminophen appears to increase hepatic NO, acting via mechanisms other than iNOS (Jaeschke & Bajt, 2006). Our study of the role of NO was limited, since NO levels and NOS activities were not measured. Nevertheless, undetectable iNOS and very low levels of eNOS mRNA in all experimental groups may suggest that excessive NO production is not a main mechanism leading to high peroxynitrite levels in our model.

Another potential mechanism of an increase in peroxynitrite is GSH depletion. Specific depletion of mitochondrial GSH causes accumulation of ROS in hepatocytes exposed to hypoxia in vitro, leading to cell death (Lluis et al. 2005). In the present study, we have shown that, while CIH and APAP alone do not affect hepatic GSH levels, a combination of both insults leads to GSH depletion. Glutathione scavenges peroxynitrite, decomposing it to NO (Halliwell & Gutteridge, 2007). Therefore, depletion of GSH by a combination of CIH and APAP can selectively increase peroxynitrite levels, causing mitochondrial damage, liver injury and hepatic necrosis.

Chronic intermittent hypoxia, APAP and hepatic inflammation

In the absence of APAP, exposure to CIH for 4 weeks caused an increase in serum AST, whereas the elevations in serum ALT and hepatocellular swelling in histological sections that were previously observed after exposure to CIH for 12 weeks (Savransky et al. 2007c), were not detected. Similar to the previous report (Savransky et al. 2007c), CIH alone did not induce significant inflammation in the liver of lean C57BL/6J mice. In contrast, in mice with diet-induced hepatic steatosis, CIH caused lobular inflammation with increased levels of TNF-α, IL-6, interleukin-1β and the chemokine MIP-2 (Savransky et al. 2007a). These data suggest that CIH acts as a ‘second hit’, causing hepatitis only in the presence of the primary insult. In the present study, CIH again acts as a powerful ‘second hit’ in APAP-induced liver injury. Acetaminophen alone significantly increased levels of IL-6 and TNF-α, consistent with previous reports (Jaeschke, 2005), but MCP-1 and MIP-2 levels rose only in the presence of CIH (Fig. 5). Tunour necrosis factor-α induces the transcription of chemokines (Bataller & Brenner, 2005; Laurens et al. 2005), but ROS may be required for induction to occur (Chen et al. 2004). We speculate that peroxynitrite up-regulated hepatic MCP-1 and MIP-2 synthesis during combined exposure to CIH and APAP. An increase in these chemotactic factors would then promote liver inflammation and injury.

Nitrosative stress is not the only mechanism of hepatic inflammation in our model. Aceaminophen increases apoptosis of hepatocytes by up-regulating apoptosis signal-regulating kinase 1 and c-Jun N-terminal kinase (Gunawan et al. 2006; Nakagawa et al. 2008), independently of peroxynitrite. Chronic intermittent hypoxia induces apoptosis in the brain and myocardium owing to a direct effect of superoxide (Gozal et al. 2001; Shan et al. 2007; Chen et al. 2008). Our data reveal that the two noxious stimuli do not affect hepatic apoptosis when administered separately. In contrast, a combination of CIH and APAP leads to a marked increase in apoptosis in the liver, which may contribute to hepatic inflammation and necrosis, independently of peroxynitrite.

Clinical implications

Our data have significant clinical implications. Obstructive sleep apnoea directly predisposes to several conditions associated with chronic pain (Loh et al. 1999; Goder et al. 2003; Gold et al. 2003, 2004). In addition, OSA is highly prevalent in obese individuals (Punjabi et al. 2002), who frequently suffer from chronic musculoskeletal pain (Janke et al. 2007; Anandacoomarasamy et al. 2008). Acetaminophen is one of the most common analgesics, and chronic treatment with APAP is frequently recommended in chronic pain syndromes associated with OSA and obesity (Felson et al. 2000; Sarzi-Puttini et al. 2005; Bennett et al. 2003). Hence, CIH of OSA and chronic APAP use may frequently coexist in clinical practice. In addition, OSA and obesity are associated with fatty liver disease (Nomura et al. 1988; Bellentani et al. 2000; Browning et al. 2004; Tanne et al. 2005; Tatsumi & Saibara, 2005; Singh et al. 2005; Jouet et al. 2007), which may lead to increased generation of ROS, depletion of GSH and augmented APAP toxicity (Laurent et al. 2004; Carmiel-Haggai et al. 2005). One limitation of our study is that the APAP dose administered to mice was two- to threefold higher than recommended for humans. Nevertheless, our data suggest that patients with OSA may be uniquely vulnerable to the hepatic toxicity of APAP, inviting future clinical investigation.

Conclusions

Chronic intermittent hypoxia greatly enhances hepatic toxicity of chronic APAP administration, resulting in liver necrosis and inflammation. The mechanism of injury appears to be related to the formation of peroxynitrite, depletion of GSH stores and apoptosis of hepatocytes.

References

  1. Top of page
  2. Abstract
  3. Methods
  4. Results
  5. Discussion
  6. References
  7. Appendix
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Appendix

  1. Top of page
  2. Abstract
  3. Methods
  4. Results
  5. Discussion
  6. References
  7. Appendix

Acknowledgements

This work was supported by National Institute of Health grants HL68715 and HL80105, and American Heart Association grant 0765293U to Dr Vsevolod Y. Polotsky, DA016370 to Dr Michael S. Torbenson, Pilot and Feasibility Grant of the Clinical Nutrition Research Unit (DK72488) to Dr Vladimir Savransky, NIH T32 HL07534 and American Lung Association-National Sleep Foundation Pickwick Fellowship SF-78568-N to Dr Jonathan Jun, American Heart Association Mid-Atlantic Affiliate Postdoctoral Fellowship 0625514U to Dr Jianguo Li and Research Fellowship grant RE 2842/1-1 of the German Research Foundation (DFG) to Dr Christian Reinke.