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

TOXICOLOGICAL SCIENCES 76, 376 –383 (2003) DOI: 10.1093/toxsci/kfg234 Promyelocytic HL60 Cells Express NADPH Oxidase and Are Excellent Targets in a Rapid Spectrophotometric Microplate Assay for Extracellular Superoxide Olga Teufelhofer,* Rosa-Maria Weiss,† Wolfram Parzefall,* Rolf Schulte-Hermann,* Michael Micksche,† Walter Berger,† and Leonilla Elbling† ,1 Institute of Cancer Research, *Division Oncological Toxicology and †Division of Applied and Experimental Oncology, University of Vienna, 1090 Vienna, Austria Received July 23, 2003; accepted August 28, 2003 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 that is frequently used for the 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 nondifferentiated HL60 cells contain NADPH oxidase and can be used as a model for the assessment of oxidant as well as antioxidant compounds. The expression of the multicomponent NADPH oxidase was demonstrated in nondifferentiated HL60 cells at the molecular level by detection of the mRNAs of the components gp91phox, p47phox, and p67phox as well as functionally by phorbol 12-myristate-13-acetate (PMA)stimulated generation of superoxide, which was susceptible to inhibition by diphenyleneiodonium. The functional assay was performed using the cells in a log growth phase by adapting a standard microplate assay based on the classic superoxide dismutase-inhibitable reduction of cytochrome c. Validation of the microplate assay was carried out both with nonadherent 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), nonoxidant (4alpha-PMA, piracetam), and antioxidant (alpha-tocopherol, ascorbic acid) activity. In summary, we established a highly specific, reproducible and— with the aid of the nondifferentiated HL60 cell line—time-saving superoxide microplate assay as a valuable tool for the rapid screening of compounds for oxidative and antioxidative activity. Key Words: promyelocytic HL60; NADPH oxidase; superoxide microplate assay; PMA; prooxidants; antioxidants. The multicomponent NADPH oxidase of activated phagocytes has been found to be the most important enzyme involved in the generation of reactive oxygen species (ROS) and oxida1 To whom correspondence should be addressed. Fax: (043) 01 427765196. E-mail: . Toxicological Sciences 76(2), © Society of Toxicology 2003; all rights reserved. tive stress (Babior, 1999; Forman et al., 2001). Upon stimulation, phagocytes synthesize ROS from a superoxide, which is formed as the earliest product of an oxidative burst by NADPH oxidase and represents a precursor of a vast assortment of ROS (Babior, 1999). Free radicals play an important role in host defenses against pathogens but also can cause tissue damage and thus are considered to be involved in the ageing process and pathogenesis of several diseases (Droge, 2002) as well as in mutagenesis and carcinogenesis (Sekiguchi et al., 2002). The identification of oxidant and antioxidant compounds is important for predicting and reducing health risks. As most of the superoxide is released to the outside of the cell, the assessment of oxidative stress is commonly performed by assays for extracellular superoxide, although methods for superoxide detection inside cells are also available (Tarpey et al., 2001). Extracellular superoxide has been determined in isolated supernatants of activated cells or, using plate reader assays, in supernatants with the cells left on the plate. Current detection methods of extracellular superoxide utilize the reduction of reactive compounds or chemiluminescence reactions due to the ability of ROS formation by an oxidation product (Tarpey et al., 2001). The latter method has been criticized due to caveats in the use of small molecules that may generate the same ROS intended to be measured (Forman et al., 2001). Plate reader assays (Catino et al., 1988; Cerasoli et al., 1988; Leslie, 1987; Madesh et al., 1997; Quick et al., 2000) have been developed on the basis of superoxide-induced reduction of exogenous ferricytochrome c (Pick et al., 1981). The specificity of this method is checked by the addition of a superoxide dismutase (SOD), which serves to confirm generated superoxide as an electron donor (Crapo et al., 1978). The use of an SOD provides one of the most reliable detection methods of superoxide generation (Tarpey et al., 2001). Targets used in plate reader assays were cell homogenates (Quick et al., 2000) or freshly prepared adherent exudate cells (Pick et al., 1981), as well as polymorphonuclear leukocytes (Cerasoli et al., 1988; Leslie, 1987), all of which are not really suitable for large-scale applications. Leukemia cell lines would allow large-scale drug 376 HL60 SUPEROXIDE MICROPLATE ASSAY screening and assessment of potential oxidative risk; however, they have been induced to differentiate to a phagocytic phenotype before use (Birnie, 1988, Catino et al., 1988), a process requiring several days. Moreover, variations in the differentiation states of cells as well as the potential toxicity of differentiation-inducing agents may significantly interfere with the reliability and reproducibility of the assay. In this study we aimed to develop a simplified plate reader assay for rapid superoxide screening and to use the standard HL60 leukemia cell line at the promyelocyte state. We confirmed these cells’ suitability by demonstrating the expression of NADPH oxidase representing the main pathway of ROS generation. In phagocytic cells, this enzyme complex catalyzes the NADPH-dependent reduction of the oxygen molecule to the superoxide anion. It consists of the proteins gp91phox and p22phox (flavocytochrome b558) anchored in the plasma membrane and the water-soluble cytosolic proteins p67phox, p47phox, p40phox, and Rac, which, upon stimulation, assemble with the flavocytochrome b558 to the active multienzyme complex (Babior, 1999; Vignais, 2002). Currently, increasing evidence suggests a more general importance of NADPH oxidase activity since various nonphagocytic cells may also contain NADPH oxidase or other oxidases of gp91, p47phox, and p67phox homologs (Banfi et al., 2001; Edens et al., 2001). Nonphagocyte NADPH oxidase has been proposed to play important roles in various signaling events and other physiological processes. Furthermore, the generation of an oxidative burst as well as highly reactive oxygen species, although at much lower levels, has been demonstrated in nonimmune cells (Droge, 2002; Lambeth, 2002). Published data on NADPH oxidase expression and superoxide (...truncated)