Acetaminophen decreases intracellular glutathione levels and modulates cytokine production in human alveolar macrophages and type II pneumocytes in vitro

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Abstract

Recent epidemiological observations suggest that acetaminophen (paracetamol) may contribute to asthma morbidity. Impaired endogenous antioxidant defences may have a role in the pathogenesis of a number of inflammatory pulmonary diseases, including asthma. We studied the effect of acetaminophen on the intracellular level of reduced glutathione (GSH) with and without inhibitors of cytochrome P450 or prostaglandin H synthetase, and TNF-α, IL-6 and IL-8 protein production in human alveolar macrophages and type II pneumocytes in vitro. Following a 20 h incubation with acetaminophen, cytotoxicity was apparent from ≥5 and ≥10 mM in macrophages and type II pneumocytes, respectively. A time- and concentration-dependent decrease of intracellular GSH occurred after acetaminophen (0.05–1 mM) exposure (1–4 h) in pulmonary macrophages (up to 53%) and type II pneumocytes (up to 34%). Diethyldithiocarbamic acid, potassium ethyl xanthate, and indomethacin decreased significantly acetaminophen-induced GSH depletion in the two cell types tested, suggesting the involvement of cytochrome P450 (mainly CYP2E1) and/or prostaglandin H synthetase. In macrophages, acetaminophen decreased the secretion of TNF-α (at 4 and 24 h, concentration-related) and IL-6 (at 24 h, at 0.1 mM), and did not affect significantly IL-8 production. These in vitro observations demonstrate that clinically relevant concentrations of acetaminophen decreased: (i) intracellular GSH in human pulmonary macrophages and type II pneumocytes and (ii) the secretion of TNF-α and possibly IL-6 by human pulmonary macrophages. These findings provide experimental plausibility to the challenging observations that frequent use of APAP may be a risk factor for asthma morbidity.

Section snippets

Chemicals and materials

Acetaminophen (APAP, paracetamol), trypsin type I (EC 3.4.214), indomethacin, diethyldithiocarbamic acid, GSH, metaphosphoric acid, bovine serum albumin, EDTA, 3-[4,5 dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT), DMSO, o-phthaldialdehyde, and glutaraldehyde were purchased from Sigma Chemical Co. Ltd. Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS), penicillin–streptomycin solution (10,000 U/ml and 10,000 μg/ml, respectively), amphotericin B (250 μg/ml) and l

Morphological characterisation of the pulmonary macrophages

The morphological features of the cells, obtained after slicing and washing lung tissue specimens, were assessed by electron microscopy in order to verify whether these free cells were indeed alveolar macrophages. The excellent ultrastructural preservation of the cells was consistent with the estimates of viability. At least 90% of the macrophages in these pellets exhibited the ultrastructure of alveolar macrophages (Fig. 1A). Most of these cells had a long axis >10 μm and an eccentric,

Discussion

These studies demonstrate, for the first time, effects of APAP on human pulmonary cells in vitro. APAP decreased (i) intracellular GSH in pulmonary macrophages and type II pneumocytes, and (ii) TNF-α and possibly IL-6 protein secretion by macrophages. These effects were observed at subtoxic and clinically relevant concentrations. The concentrations of APAP that are reached in the pulmonary interstitial and epithelial lining fluid are not known, but as APAP is rapidly and uniformly distributed

Acknowledgments

This work was supported by a fellowship from the European Respiratory Society to S. Dimova and partly by INCO/Copernicus (EU) (IC15-CT96-0314). We are grateful to Judy McWilliam for the preparation of samples for electron microscopy.

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