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NAD(P)H oxidases (NOXs) are a family of enzymes catalyzing the univalent reduction of oxygen to produce the superoxide anion radical, which in turn can be converted in other reactive oxygen species (ROS) and may participate to the formation of reactive nitrogen derivatives, such as peroxynitrite. By virtue of their activity, NOXs may represent a double-edged sword for the organism's homeostasis. On one hand ROS participate in host defence by killing invading microbes and may regulate several important physiological functions, such as cell signalling, regulation of cell growth and differentiation, oxygen sensing, angiogenesis, fertilization and control of vascular tone. On the other hand ROS may play an important role in pathological processes such as hypertension, atherosclerosis, diabetes, cancer, ischemia/reperfusion injury, neurodegenerative diseases. Many roles suggested for NOXs in various tissues and physiopathological situations have been inferred by the in vitro and in vivo effects of several NOX inhibitors. In particular, most studies are based on the use of two compounds, diphenyleneiodonium and apocynin. Aim of this review is to describe the main features of these two compounds, to show that they cannot be used as specific NOX inhibitors and to solicit researchers to find other tools for investigating the role of NOXs.

Diphenyleneiodonium (DPI) and the structurally related compound diphenyliodonium (DIP) are widely used as inhibitors of flavoenzymes, particularly NADPH oxidase. Here we report further evidence that DPI and DIP are not specific flavin binders. A 3-h incubation of N11 glial cells with DPI significantly inhibited in a dosedependent way both the pentose phosphate pathway and the tricarboxylic acid cycle. In parallel, we observed a dose-dependent increase of reactive oxygen species generation and lipoperoxidation and increased leakage of lactate dehydrogenase activity in the extracellular medium. The glutathione/glutathione disulfide ratio decreased, whereas the efflux of glutathione out of the cells increased. This suggests that DPI causes an augmented oxidative stress and exerts a cytotoxic effect in N11 cells. Indeed, the cells were protected from these events when loaded with glutathione. Similar results were observed using DIP instead of DPI and also in other cell types. We suggest that the DPI-elicited inhibition of the pentose phosphate pathway and tricarboxylic acid cycle may be mediated by the blockade of several NAD(P)-dependent enzymes, such as glucose 6-phosphate dehydrogenase, glyceraldehyde 3-phosphate dehydrogenase, and lactate dehydrogenase. In light of these results, we think that some effects of DPI or DIP in in vitro and in vivo experimental models should be interpreted with caution. Diphenyleneiodonium (DPI) 1 and the structurally related compound diphenyliodonium (DIP) are widely used as uncompetitive inhibitors of flavoenzymes. Firstly identified as a hypoglycemic agent able to block gluconeogenesis and respiration in rat liver (1), DPI has been subsequently shown to inhibit the activity of NADH:ubiquinone oxidoreductase (2, 3), NADPH oxidase (4 -6), nitric-oxide synthase (7), xanthine oxidase (6), and NADPH cytochrome P450 oxidoreductase (8). DPI and other iodonium derivatives have been shown to react via a radical mechanism, whereby an electron is abstracted from FAD or FMN to form a radical, which then adds back to the flavin to form covalent, phenylated adducts (9). Also, heme groups, such as the heme b of NADPH oxidase, have been found to react with DPI and DIP (10).In most experimental works of the last years, DPI or DIP have been used as inhibitors of NADPH oxidase. NADPH oxidases are a group of plasma membrane-associated enzymes found in a variety of cells of mesodermal origin. The most thoroughly studied is the leukocyte isoform, which catalyzes the production of superoxide (O 2 . ) by the one-electron reduction of oxygen, using NADPH as the reducing agent (11). The O 2 .generated by NADPH oxidase serves as the starting material for the production of a vast assortment of reactive oxidants used by phagocytes to kill invading microorganisms or tumor cells (11). A low activity NADPH oxidase is present in a variety of nonphagocytic cells, wherein this enzyme is a source of second messengers. It has been postulated, for instance, that the O 2 . generated by the aorta functions as a blood ...

BackgroundInvasive micropapillary carcinoma (IMPC) of the breast is a distinct and aggressive variant of luminal type B breast cancer that does not respond to neoadjuvant chemotherapy. It is characterized by small pseudopapillary clusters of cancer cells with inverted cell polarity. To investigate whether hypoxia-inducible factor-1 (HIF-1) activation may be related to the drug resistance described in this tumor, we used MCF7 cancer cells cultured as 3-D spheroids, which morphologically simulate IMPC cell clusters.MethodsHIF-1 activation was measured by EMSA and ELISA in MCF7 3-D spheroids and MCF7 monolayers. Binding of HIF-1α to MDR-1 gene promoter and modulation of P-glycoprotein (Pgp) expression was evaluated by ChIP assay and FACS analysis, respectively. Intracellular doxorubicin retention was measured by spectrofluorimetric assay and drug cytotoxicity by annexin V-FITC measurement and caspase activity assay.ResultsIn MCF7 3-D spheroids HIF-1 was activated and recruited to participate to the transcriptional activity of MDR-1 gene, coding for Pgp. In addition, Pgp expression on the surface of cells obtained from 3-D spheroids was increased. MCF7 3-D spheroids accumulate less doxorubicin and are less sensitive to its cytotoxic effects than MCF7 cells cultured as monolayer. Finally, HIF-1α inhibition either by incubating cells with 3-(5'-hydroxymethyl-2'-furyl)-1-benzylindazole (a widely used HIF-1α inhibitor) or by transfecting cells with specific siRNA for HIF-1α significantly decreased the expression of Pgp on the surface of cells and increased the intracellular doxorubicin accumulation in MCF7 3-D spheroids.ConclusionsMCF7 breast cancer cells cultured as 3-D spheroids are resistant to doxorubicin and this resistance is associated with an increased Pgp expression in the plasma membrane via activation of HIF-1. The same mechanism may be suggested for IMPC drug resistance.

"Vitreous silica" is a particular form of amorphous silica, much neglected in experimental studies on silica toxicity. In spite of the incorrect term "quartz glass", often employed, this material is fully amorphous. When reduced in powdered form by grinding, the particulate appears most close to workplace quartz dust but, opposite to quartz, is not crystalline. As silicosis and lung cancer are also found among workers exposed to "quartz glass", the question arises of whether crystallinity is the prerequisite feature that makes a silica dust toxic. We compare here the behavior of comminuted quartz, vitreous silica, and monodispersed silica spheres, as it concerns surface reactivity and cellular responses involved in the accepted mechanisms of silica toxicity. Care was taken to choose samples of extreme purity, to avoid any effect due to trace contaminants. Quartz and vitreous silica, opposite to silica spheres, show irregular particles with sharp edges, stable surface radicals, and sustained release of HO(*) radicals via a Fenton-like mechanism. The evolution of the heat of adsorption of water as a function of coverage shows with quartz and vitreous silica a similar pattern of strong hydrophilic sites, nearly absent on the other silica specimen. When tested on a macrophage cell line (MH-S), vitreous silica and pure quartz, but not the monodispersed silica spheres, showed a remarkable potency in cytotoxicity, nitric oxide synthase activation and release of nitrite, and tumor necrosis factor-alpha production, suggesting a common behavior in inducing an oxidative stress. All of the above features appear to indicate that crystallinity might not be a necessary prerequisite to make a silica particle toxic.