Environmental Health Perspectives 105, Supplement 5, September 1997: Article by Borm et al.

Environmental Health Perspectives 105, Supplement 5, September 1997

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Neutrophils Amplify the Formation of DNA Adducts by Benzo[a]pyrene in Lung Target Cells

Paul J.A. Borm, Ad M. Knaapen, Roel P.F. Schins, Roger W.L. Godschalk, and Frederik-Jan Van Schooten

Department of Health Risk Analysis and Toxicology, Maastricht University, Maastricht, The Netherlands

Abstract
Inflammatory cells and their reactive oxygen metabolites can cause mutagenic effects in lung cells. The purpose of this study was to investigate the ability of activated neutrophils to modulate DNA binding of benzo[a]pyrene (B[a]P), a known carcinogen, in lung target cells. Equivalent numbers of rat lung epithelial cells (RLE-6TN cell line) and freshly isolated human blood neutrophils (PMN) were coincubated in vitro for 2 hr after addition of benzo[a]pyrene (0.5 µM) or two of its trans-diol metabolites, with or without stimulation with phorbol myristate acetate (PMA). DNA adducts of B[a]P-metabolites were determined in target cells using ³² P-postlabeling; oxidative DNA damage (7-hydro-8-oxo-2´-deoxyguanosine [8-oxodG]) was evaluated by high performance liquid chromatography with electrochemical detection. Increased DNA adducts were observed in lung cells coincubated with polymorphonuclear leukocytes (PMN). Activation of PMN with PMA, or addition of more activated PMN in relation to the number of lung cells, further increased the number of adducts, the latter in a dose-response manner. Incubation with B[a]P-4,5-diol did not result in any adduct formation, while B[a]P-7,8-diol led to a significant number of adducts. Moreover, PMA-activated PMN strongly enhanced adduct formation by B[a]P-7,8-diol, but not 8-oxodG, in lung cells. The addition of antioxidants to the coincubations significantly reduced the number of adducts. Results suggest that an inflammatory response in the lung may increase the biologically effective dose of polycyclic aromatic hydrocarbons (PAHs), and may be relevant to data interpretation and risk assessment of PAH-containing particulates. -- Environ Health Perspect 105(Suppl 5):1089-1093 (1997)

Key words : inflammation, lung, DNA adducts, benzo[a]pyrene, coincubations, neutrophils

This paper is based on a presentation at The Sixth International Meeting on the Toxicology of Natural and Man-Made Fibrous and Non-Fibrous Particles held 15-18 September 1996 in Lake Placid, New York. Manuscript received at EHP 26 March 1997; accepted 19 June 1997.

We acknowledge K.E. Driscoll (Procter & Gamble) for providing RLE-6TN cells.

Address correspondence to Dr. P.J.A. Borm, Associate Professor in Toxicology and Industrial Hygiene, Department of Health Risk Analysis and Toxicology, P.O. Box 616, 6200 MD Maastricht, The Netherlands. Telephone: 31 43 3881097. Fax: 31 43 3670924. E-mail: p.borm@grat.unimaas.nl

Abbreviations used: ATP, adenosine triphosphate; B[a]P, benzo[a]pyrene; BPDE, benzo[a]pyrene diolepoxide; dAp, deoxyadenosine-3´-monophosphate; dG, deoxyguanosine; DMSO, dimethylsulfoxide; FCS, fetal calf serum; HBSS, Hanks balanced salt solution; HPLC/ECD, high performance liquid chromatography/electrochemical detection; MPO, myeloperoxidase; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; NP1, nuclease P1; nt, nucleotides; 8-oxodG, 7-hydro-8-oxo-2´-deoxyguanosine; PAH, polycyclic aromatic hydrocarbon; PBS, phosphate-buffered saline; PEI, polyethylenimine; PMA, phorbol-myristate-acetate; PMN, polymorphonuclear leukocytes; PNK, polynucleotide kinase; RLE, rat lung epithelial; ROS, reactive oxygen species; SOD, superoxide dismutase; TPA, 12-O-tetradecanoylphorbol-13-acetate.

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Last Update: October 16, 1997