Elsevier

Biochemical Pharmacology

Volume 59, Issue 11, 1 June 2000, Pages 1467-1475
Biochemical Pharmacology

Cardiovascular and Pulmonary Pharmacology
Paracetamol (acetaminophen) cytotoxicity in rat type II pneumocytes and alveolar macrophages In Vitro

https://doi.org/10.1016/S0006-2952(00)00257-4Get rights and content

Abstract

Paracetamol (acetaminophen, APAP) liver and kidney cytotoxicity is associated with bioactivation by P450 and/or prostaglandin H synthetase (PGHS) to a reactive metabolite, which depletes GSH, covalently binds to proteins, and leads to oxidative stress. Although APAP may also damage the lung, little is known about the mechanism by which this occurs. We studied the in vitro toxicity of APAP and its effect on the intracellular GSH level in rat type II pneumocytes (freshly isolated or 24-hr-old) and alveolar macrophages. Cytotoxicity was evaluated by changes in membrane integrity (lactate dehydrogenase, [LDH] assay) as well as by mitochondrial metabolic activity (reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, MTT), following a 20-hr incubation with APAP (1–20 mM). APAP caused a concentration-related decrease in MTT reduction and LDH retention in the fraction of attached cells, which was associated with an increase in LDH activity in the medium and in the fraction of non-attached cells. The order of susceptibility was: freshly isolated type II pneumocytes > alveolar macrophages > 24-hr-old type II pneumocytes. A time- and concentration-dependent decrease in intracellular GSH occurred in freshly isolated type II pneumocytes and alveolar macrophages exposed to subtoxic (≤ 1 mM) APAP concentrations. In 24-hr-old type II pneumocytes, there were no changes in intracellular GSH concentration after APAP exposure. Potassium ethyl xanthate (a P450 inhibitor) and indomethacin (a PGHS inhibitor) significantly decreased APAP-induced GSH depletion in freshly isolated type II pneumocytes and alveolar macrophages, suggesting that P450 and/or PGHS are involved in APAP bioactivation in these cells.

Section snippets

Chemicals and materials

Acetaminophen, indomethacin, trypsin type I (EC 3.4.21.4, Cat. No. T-8003), GSH, pyruvic acid, NADH, metaphosphoric acid (HPO3), bovine serum albumin, Percoll, EDTA, MTT, DMSO, and o-phthaldialdehyde were purchased from Sigma. DMEM, FBS, penicillin–streptomycin solution (10,000 U/mL and 10,000 μg/mL, respectively), fungizone (250 μg/mL), and l-glutamine (200 mM) were obtained from GIBCO. DNase I (EC 3.1.21.1) was purchased from Boehringer Mannheim GmbH. Protein assay dye solution was obtained

In vitro toxicity of APAP

After 20-hr incubation, APAP caused a concentration-related decrease in MTT reduction in rat type II pneumocytes (freshly isolated and 24-hr-old) and alveolar macrophages (Fig. 1). The reduction of MTT was significantly decreased compared to control at ≥ 5 mM APAP in freshly isolated type II pneumocytes and alveolar macrophages, and at ≥ 10 mM in 24-hr-old type II cells. APAP caused a significant decrease in LDH retention in the fraction of attached cells in freshly isolated type II

Discussion

These studies demonstrated that rat type II pneumocytes and alveolar macrophages are sensitive to APAP-induced toxicity in vitro in a concentration-dependent manner, with the order of susceptibility being as follows: freshly isolated type II pneumocytes > alveolar macrophages > 24-hr-old type II pneumocytes. The concentrations that caused cytotoxicity may appear quite high, since APAP concentrations of ≥ 5 mM were needed to produce cytotoxicity in freshly isolated type II pneumocytes and

Acknowledgements

This work was supported by the Belgian Office for Scientific, Technical, and Cultural Affairs and partly by INCO/Copernicus (EU) (IC15-CT96-0314).

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