Experimental Cell Research 179 (1988) 581-589 SHORT NOTE Microinjection of Antibodies against Superoxide Dismutase and Glutathione Peroxidase C. MICHIELS,’ M. RAES, M.-D. ZACHARY, E. DELAIVE, and J. REMACLE Laboratoire de Biochimie Cellulaire, Facult& Universitaires ND de la Paix, rue de Bruxelles, 61, B-5000 Namur, Belgium Antibodies were prepared against glutathione peroxidase, superoxide dismutase, and catalase. Inhibition of the enzyme activity was obtained with anti-Gpx and anti-SOD antibodies but not with anti-CAT antibodies. The antibodies were then injected into human tibroblasts and bovine chondrocytes in culture either under normal conditions or under 1 atm of oxygen. The injected anti-Gpx and anti-SOD antibodies increased the mortality rate of the libroblasts incubated under 1 atm of oxygen. However, when cells were incubated under normal atmosphere, anti-Gpx antibodies inhibited the division while anti-SOD antibodies increased this capacity. Anti-Gpx antibodies injected into chondrocytes de- creased their viability. Injection of control antiserum had no effect. These data stress the primary importance of Gpx as antioxidant under all conditions and the relative efftciency of SOD according to the balance between the radical production and the activity of the other antioxidant systems. 0 1988 Academic PKSS, IIIC. The balance between the production of oxidants and their inactivation by antioxidant defenses is critical for the maintenance of cellular integrity [I]. The destruction of the free radicals is performed by a series of systems including antioxidant molecules such as a-tocopherol, ascorbic acid, glutathione [2], and antioxidant enzymes: superoxide dismutase (SOD), catalase (CAT), and gluta- thione peroxidase (Gpx). Superoxide dismutase discovered by McCord and Fridovich [3] catalyzes the dismutation of superoxide anion to hydrogen peroxide and oxygen [4]. Eukaryot- ic cells contain two different SODS: one containing copper and zinc (CuZnSOD) found in the cytosol and intermembrane space of mitochondria and one contain- ing manganese (MnSOD) located in the mitochondrial matrix [4]. The steady- state concentration of 0: in the cell is kept between lo-” and lo-l2 M [5]. Catalase, a heme-containing enzyme destroying H202, is located in peroxi- somes, cytosol [6], and mitochondrial matrix [71. The intracellular steady-state concentration of H202 is kept between lo-’ and 10m9 M [5]. Glutathione peroxidase, a selenium-dependent enzyme, discovered by Mills [8] reduces hydroperoxides and H202 using reduced glutathione as the second sub- strate 191. Therefore, the glutathione peroxidase activity is dependent on glutathi- one reductase activity as well as the availability of NADPH. The subcellular distribution of Gpx is complementary to that of catalase: two-thirds is in the cytosol and one-third is in the mitochondrial matrix [93. ’ To whom reprint requests should be addressed. 38-888342 581 Copyright @ 1988 by Academic Press, Inc. A0 rights of reproduction in any form reserved 0014-4827/88 $03.00