Acetaminophen-induced Mitochondrial Oxidative Stress in Murine J774.2 Monocyte Macrophages

Abstract

The cytotoxic potential of an antipyretic and analgesic drug, acetaminophen (APAP), was evaluated in mouse J774.2 monocyte macrophages. The cytotoxicity of APAP was evaluated by MTT cell viability and apoptosis assays. Based on the cell viability and apoptosis assays, further experiments were designed with a low (1 µmol/ml) and a high (10 µmol/ml) dose treatment of APAP in J774.2 cells. Mitochondrial oxidative stress, reactive oxygen species (ROS), mitochondrial glutathione (GSH) metabolism, lipid and protein peroxidation were measured in the drug treated cells. An increase in mitochondrial oxidative stress and ROS production was observed. A decrease in the mitochondrial GSH pool, accompanied by an increase in lipid and protein peroxidation appeared to be the main cause of mitochondrial oxidative stress. GSH pool and GSH metabolizing enzymes were differentially affected in the mitochondria and extramitochondrial compartments. Increased nuclear translocation of NF-kB-p65, a marker of redox metabolism was also observed in the drug treated cells. In addition, we have demonstrated, for the first time that the mitochondrial aconitase enzyme is a potential ROS-sensitive target in J774.2 cells, which might be used as a marker for APAP-induced cytotoxicity. These results have clearly suggested that APAP induced cytotoxicity in macrophages is mediated by increased mitochondrial oxidative stress and altered redox metabolism. This might have implications in determining the role of circulating macrophages against APAP induced toxicity and cellular defenses in tissues.

Comparative evaluation of N-acetylcysteine and N-acetylcysteineamide in acetaminophen-induced hepatotoxicity in human hepatoma HepaRG cells

“…Decreased activity of GR seen in our experiments upon APAP treatment supports our hypothesis of GSH depletion as a key event in APAP-induced hepatotoxicity. Our results are in accordance with Al-Belooshi et al 62 who reported a decrease in mitochondrial GR after APAP treatment.…”

“…Levels of MDA, a lipid peroxidation by-product, provided a suitable index for measurement of lipid peroxidation in vivo. In accordance with previous studies, 60,62,65,68,[74][75][76] we observed increased levels of MDA after 24 h of APAP treatment. In general, once toxic lipid peroxides are formed by free radicals, GPx attempts to eliminate them at the expense of GSH.…”

“…Our data are in agreement with other studies that have reported an increase in the production of ROS upon APAP treatment. 56,62 In addition, Bajt et al 56 showed that ROS was produced as a consequence of decreased GSH levels. However, pretreatment with NAC/NACA significantly decreased the ROS ( Figure 5…”

Differential Cytotoxicity of Acetaminophen in Mouse Macrophage J774.2 and Human Hepatoma HepG2 Cells: Protection by Diallyl Sulfide

“…Cells were treated with APAP (10 μmol/ml) for 18 hours after treatment with or without 200 μM DAS for 24 h. After the desired time of treatment, cells were harvested, washed with PBS (pH 7.4) and homogenized in H-medium buffer (70 mM sucrose, 220 mM mannitol, 2.5 mM HEPES, 2 mM EDTA, and 0.1 mM phenylmethylsulfonylfluoride, pH7.4) at 4°C. Mitochondria and postmitochondrial (PMS) fractions were prepared by centrifugation and the purity of the isolated fractions for cross contamination was checked as described before [ 24 – 28 ]. Control cells were treated with vehicle alone.…”

“…Both cytotoxic and cytoprotective effects of macrophages have been reported in APAP-induced toxicity [ 19 , 22 – 23 ]. Our previous study on J774.2 macrophages demonstrated that APAP induces cytotoxicity and apoptosis by increasing ROS production, depletion of GSH pool, increase in oxidative stress and mitochondrial dysfunction [ 24 – 25 ]. Using macrophages and HepG2 cells as in vitro models, we have recently reported that aspirin treatment also induces oxidative stress and mitochondrial dysfunction, albeit at different levels [ 26 – 29 ].…”