182 murine adenocarcinoma (MAC) 16 present with solid tumour growth and cachexia [4], as have been shown in rats bearing the Yoshida AH-130 hepatoma [5], character- istic of the human cancer cachectic condition. An abundance of evidence exists for the induction of the ubiquitin (Ub)-proteasome proteolytic pathway in the development of cancer cachexia; however, evidence of protein oxidation in cancer cachectic patients suggests the involvement of reactive oxygen species (ROS) [6]. Studies in both humans and animals with cancer cachexia have shown the presence of oxidised proteins [6]. Furthermore, cancer patients have been shown to present with elevated levels of serum ROS and lower antioxidant levels [7], indicative of a pro-oxidative shift. Similarly, increased levels of ROS have been shown in cachectic tumour- bearing rats, with no compensatory response from antioxidant enzymes [5]. Although the catabolic mechanism leading to the severe muscle wasting associated with cancer develop- ment remains relatively unknown, there is evidence to suggest a role for ROS [7]. Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX) is a multicomponent enzyme that when activated catalyzes the production of superoxide anion (O2−). The NOX2 enzyme consists of five subunits segregated into membrane bound, NOX2 (gp91phox) and p22phox, and cytosolic, p67phox, p47phox and p40phox, units. On stimulation, the protein components of the NOX2 enzyme assemble, forming the active oxidase and the production of O2− [8]. It has been apparent in recent years that non-phagocytes possess homologues to NOX2, and that these NOX enzymes may activate in a similar manner to the well-established phagocyte NOX2 enzyme [9]. Currently, there are five known NOX homologues (NOX1-5) found to function in a variety of tissues, with the expression of both NOX2 and NOX4 enzyme subunits in skeletal muscle tissue, although its function in this particular cell system remains undefined [10]. Despite the unknown functionality of the NOX enzymes in skeletal muscle tissue, O2− generated from NOX has been impli- cated in progressive skeletal muscle damage [11]. The abundance of evidence for NOX enzyme-generated O2− in disease [11], establishes its potential role in the catabolic skeletal muscle wasting in cancer cachexia. It is well-known that ROS are generated in skeletal muscle as an essential by-product of cellular metabolism and oxidative enzymes [12]. Due to the high metabolic activity and oxidative capacity of skeletal muscle, the antioxidant system is a crucial component for the mainte- nance of cellular oxidative homeostasis [13]. With the knowledge that ROS are likely catabolic factors in the development of cancer cachexia, antioxidant enzymes are of equal importance for their crucial role in the maintenance of cellular oxidative homeostasis and the protection against J Cachexia Sarcopenia Muscle (2011) 2:181–188 harmful ROS [13]. O2− is dismutated by the O2− specific antioxidant enzyme, superoxide dismutase (SOD). Three distinct forms of SOD exist in the intracellular cytoplasmic compartment (SOD1), mitochondria (SOD2) and extracel- lular matrix (SOD3) [14]. The SOD enzymes catalyse the conversion of O2− to H2O2, which is further converted by catalase and glutathione peroxidise (GPx) scavenging [14]. A depression in these antioxidant enzyme systems can consequently lead to an imbalance in cellular oxidation and oxidative tissue damage [13], which has been shown particularly in response to disease [5]. Endogenous anti- oxidants such as SOD and GPx are low in cancer patients presenting with high levels of ROS, indicating the importance of tight regulation from endogenous antioxidants [7]. The Ub-proteasome proteolytic pathway has been established in cancer-induced cachexia. In addition, tumour-derived factors such as TNF-α and proteolysis- inducing factor (PIF) are well recognised in the cancer cachectic condition [15]. What is yet to be elucidated, however, are the downstream mediators of this complex system, in response to tumour growth and development of cancer cachexia. A study by Russell et al. [16] proposed a cachectic pathway of increased Ub gene expression, downstream of NOX-generated ROS production, estab- lishing a potential role for the NOX enzymes in mediating skeletal muscle atrophy through the Ub-proteasome proteolytic pathway. Therefore, this study aims to investigate the O2− generating NOX and antioxidant enzyme systems in a well-established animal model of cancer cachexia. In particular, this study aims to determine O2− levels and NOX enzyme system expression (NOX2, NOX4, p22phox, p40phox, p47phox, p67phox and Rac1) in the skeletal muscle of cachectic (MAC16) tumour-bearing mice compared to non-cachectic (MAC13) tumour-bearing mice. Further- more, this study aims to determine levels of the primary antioxidants for O2− and H2O2 dismutation (SOD1, SOD2, SOD3, catalase and GPx), in the skeletal muscle of tumour- bearing cachectic mice (MAC16) compared to tumour- bearing non-cachectic (MAC13) mice. 2 Methods 2.1 Animal model of cancer-induced cachexia All experimental procedures were carried out with approval from the Victoria University Animal Ethics Committee (AEETH 07/05). Two weight-matched groups of female BALB/c nu/nu mice were maintained under controlled environmental conditions, 12-h light/dark cycle, 21°C ± 2°C, 30% humidity, in conventional cages with ad libitum access to standard chow and water throughout