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ToxSci Advance Access published online on September 26, 2003
Toxicological Sciences, doi:10.1093/toxsci/kfg234
Toxicological Sciences © Society of Toxicology 2003; all rights reserved
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Received July 23, 2003; accepted August 28, 2003
© 2003 Society of Toxicology

In Vitro Toxicology and Alternative Testing

Promyelocytic HL60 Cells Express NADPH Oxidase and Are Excellent Targets in a Rapid Spectrophotometric Microplate Assay for Extracellular Superoxide

Olga Teufelhofer 1, Rosa-Maria Weiss 2, Wolfram Parzefall 1, Rolf Schulte-Hermann 1, Michael Micksche 2, Walter Berger 2, and Leonilla Elbling 2*

1 Institute of Cancer Research, Division Oncological Toxicology, University of Vienna, 1090 Vienna, Austria
2 Institute of Cancer Research, Division of Applied and Experimental Oncology, University of Vienna, 1090 Vienna, Austria

* To whom correspondence should be addressed. E-mail: leonilla.elbling{at}univie.ac.at.


  Abstract

A great number of drugs, toxicants, and growth factors induce the generation of intermediary reactive oxygen species (ROS). The human promyelocytic leukemia HL60 cell line differentiated along the macrophage or neutrophil lineage is a model system frequently used for generation of ROS by various agents. As a primary source of ROS the superoxide anion produced by an enzymatic complex, NADPH oxidase, is well established. The present study shows that non-differentiated HL60 cells do contain NADPH oxidase and can be used as a model for the assessment of oxidant as well as antioxidant compounds. Expression of the multicomponent NADPH oxidase was demonstrated in non-differentiated HL60 cells a) at the molecular level by detection of the mRNAs of components gp91phox, p47phox, and p67phox as well as b) functionally by PMA-stimulated generation of superoxide which was susceptible to inhibition by diphenyleneiodonium. The functional assay was performed using the cells in log growth phase by adapting a standard microplate assay based upon the classic superoxide dismutase-inhibitable reduction of cytochrome c. Validation of the microplate assay was carried out both with non-adherent differentiated HL60 cells and the adherent mouse monocyte-macrophage-like RAW 264.7 cell line, as well as with various compounds of oxidant (bleomycin sulfate, cis-diammineplatinum(II), camptothecin, TNF-alpha, IL-1beta), non-oxidant (4alpha-PMA, piracetam), and antioxidant (alpha-tocopherol, ascorbic acid) activity. In summary, we established a highly specific, reproducible and - with the aid of the non-differentiated HL60 cell line - time-saving superoxide microplate assay as a valuable tool for rapid screening of compounds for oxidative and antioxidative activity.

Key Words: promyelocytic HL60, NADPH oxidase, superoxide microplate assay, PMA, prooxidants, antioxidants .


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