NADPH Oxidase Activation Is Required in Reactive Oxygen Species Generation and Cell Transformation Induced by Hexavalent Chromium

Hexavalent chromium [Cr(VI)] is a well-known human carcinogen associated with the incidence of lung cancer. Although overproduction of reactive oxygen species (ROS) has been suggested to play a major role in its carcinogenicity, the mechanisms of Cr(VI)-induced ROS production remain unclear. In this study, we investigated the role of NADPH oxidase (NOX), one of the major sources of cellular ROS, in Cr(VI)-induced oxidative stress and carcinogenesis. We found that short-term exposure to Cr(VI) (2mM) resulted in a rapid increase in ROS generation in Beas-2B cells, and concomitantly increased NOX activity and expression of NOX members (NOX1–3 and NOX5) and subunits (p22 phox , p47 phox , p40 phox , and p67 phox ). Cr(VI) also induced phosphorylation of p47 phox and membrane translocation of p47 phox and p67 phox , further confirming NOX activation. Knockdown of p47 phox with a short hairpin RNA attenuated the ROS production induced by Cr(VI). Chronic exposure (up to 3 months) to low doses of Cr(VI) (0.125, 0.25, and 0.5mM) also promoted ROS generation and the expression of NOX subunits, such as p47 phox and p67 phox , but inhibited the expression of main antioxidant enzymes, such as superoxidase dismutase (SOD) and glutathione peroxidase (GPx). Chronic Cr(VI) exposure resulted in transformation of Beas-2B cells, increasing cell proliferation, anchorage independent growth in soft agar, and forming aggressive tumors in nude mice. Stable knockdown of p47 phox or