Chronic intermittent hypoxia and acetaminophen induce synergistic liver injury in mice

Abstract

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), gamma-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.

Figure 1

The effects of chronic intermittent hypoxia (CIH) and acetaminophen (APAP) on serum enzymatic activity of (A) alanine aminotransferase (ALT), (B) aspartate aminotransferase (AST), (C) gamma glutamyl transferase (GGT) and (D) serum levels of total bilirubin. * and † denotes p < 0.05 and p < 0.001 respectively for the difference between CIH and intermittent air control; ‡ denotes p < 0.001 for the difference between APAP and placebo.

Figure 2

Liver histology in mice exposed to (A) intermittent air (IA) and placebo, (B) intermittent air (IA) and acetaminophen (APAP), (C) chronic intermittent hypoxia (CIH) and placebo, (D) chronic intermittent hypoxia (CIH) and acetaminophen (APAP). 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 exposures to IA with or without APAP (A, B) and chronic intermittent hypoxia (CIH) without APAP (C). All images H&E with original magnification of 100X.

Figure 3

TUNEL (Terminal deoxynucleotidyl Transferase Biotin-dUTP Nick End Labeling) in liver tissue of mice exposed to (A) intermittent air and placebo, (B) chronic intermittent hypoxia (CIH) and acetaminophen (APAP). White arrows point to apoptotic cells. (C) Quantification of the effects of CIH and APAP on the % of apoptotic liver cells. * denotes p < 0.01 for the difference between CIH plus APAP and all other groups. All images show TUNEL staining with original magnification of 400X.

Figure 4

The effects of chronic intermittent hypoxia (CIH) and acetaminophen (APAP) on levels of (A) reduced glutathione (GSH), (B) 3-nitrotyrosine (3-NT), and (C) malondialdehyde (MDA) in the liver. * denotes p < 0.05 for the difference between CIH and intermittent air control; † denotes p < 0.01 for the differences between CIH and intermittent air control in mice treated with APAP and between placebo and APAP in mice subjected to CIH.

Figure 5

The effects of chronic intermittent hypoxia (CIH) and acetaminophen (APAP) on levels of (A) tumor necrosis factor alpha (TNF-α), (B) interleukin 6 (IL-6), (C) monocyte chemoattractant protein-1 (MCP-1), and (D) macrophage inflammatory protein-2 (MIP-2) in the liver. * denotes p < 0.05 for the difference between CIH and intermittent air control.