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•− 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.