Introduction Metallothioneins (MT) are low-molecular weight, metal- and sulfur-rich proteins that are widely distributed in living organisms. These intracellular proteins are thought to be involved in both heavy metal detoxification and the homeo- stasis of essential trace metals, such as zinc and copper. 1–3 It has also been suggested that MT is capable of protecting cells from the damage caused by the free radicals produced in some reactions, such as, for example, the mitochondria electron transport system and phagocyte superoxide burst. 4,5 It has also been demonstrated that MT provides cells with radioresistance, 6 and that rabbit liver MT quenches the free superoxide and hydroxyl radicals produced by xanthine/ xanthine–oxidase reactions. 7 Phagocyte leucocytes reveal a mechanism through which a generation of radicals may be triggered off by several types of stimuli. During phagocytosis, these radicals are responsible for the enhanced microbiocidal activity of these cells. 8 The source of these radicals is not the mitochondria respiratory chain, but rather a membrane-associated reduced nicoti- namide adenine dinucleotide phosphate (NADPH)-oxidase. 9,10 Large amounts of superoxide (•O 2 – ) anion and hydrogen peroxide (H 2 O 2 ) 11 are released by peritoneal macrophages activated by intraperitoneal injection of thioglycollate, when stimulated with phorbol-12-myristate-13-acetate (PMA). In an earlier investigation, we reported that cadmium (Cd) modulates •O 2 – production by macrophages of the catfish Ictalurus melas activated with Concanavalin A or Saccharo- myces cerevisiae. 12 As it is also known that Cd induces MT in mouse peri- toneal macrophages, 13 the reaction mechanism of •O 2 – with Cd-MT was investigated to study the metallothionein scavenging role in living cells. Materials and Methods Macrophage harvesting and cultivation Macrophages were obtained from rat peritoneum following the method described in Robertson et al. 14 Wistar rats weighing 250–300 g were intraperitoneally injected with 29 g/L thioglycollate (Difco, Detroit, MI, USA) broth. Four days later, the animals were suppressed, and the peritoneal exudate was collected and centrifuged at 400 × g. The cell sediment was resuspended in DMEM, to which 10% FCS was added. Cells were seeded in Petri dishes to a final concentration of 5 × 10 5 cells/cm 2 . After adhesion, the resulting cultures were washed with PBS to remove non-adherent cells. 15 Macrophages (1 × 10 6 /well) were then covered with 450 μL of Kreeb’s ringer phosphate buffer (KRPGCa) (123 mmol/L NaCl, 1.23 mmol/L MgCl 2 , 4.9 mmol/L KCl and 16.7 mmol/L Na-phosphate buffer, pH 7.4), containing 5 mmol/L glucose, 0.5 mmol/L CaCl 2 and 2 mmol/L NaN 3 and supplemented with 80 μmol/L cytochrome c (Sigma, St Louis, MO, USA). 16 After 10 min incubation at 37°C (5% CO 2 ), 50 μL of PMA (Sigma) 200 nmol/L and 10 μL CdCl 2 , at various Cd concentrations, were added. After incubation at 37°C in 5% CO 2 for varying time periods (i.e. from 1 to 6 h), the medium was aspirated to determine the super- oxide ion, and cells were lysed for proteins and MT determination. Samples of untreated macrophages were used as controls. Assay for superoxide anion generation The medium, 350 μL, was aspirated from each well and diluted 1:3 with cold KRPGCa. The reduced cytochrome c was measured by analysing the difference in absorbency at 550–468 nm using a Immunology and Cell Biology (2001) 79, 251–254 Research Article Oxidative burst and metallothionein as a scavenger in macrophages P IRATO, G SANTOVITO, E PICCINNI and V ALBERGONI Department of Biology, University of Padova, Padova, Italy Summary The role of metallothionein (MT) in the scavenging of superoxide radicals (•O 2 – ) generated by macrophages has been examined. The present work has focused on the effects of added cadmium, a known inducer of MT biosynthesis, on determined amounts of superoxide radicals produced by in vitro cultured rat peritoneal macrophages on their stimulation with phorbol-12-myristate-13-acetate (PMA). The levels of superoxide radicals (•O 2 – ) have been found to decrease when cadmium was added to cells exposed to PMA. However, substantially lower levels of MT have been determined in this case compared to cells untreated with PMA. This effect could be reversed by incubation of the PMA and cadmium-treated cells with a reducing agent, 2-mercaptoethanol (2-ME). Results suggest that •O 2 – caused thiolate oxidation and subsequent metal loss, thus reducing the cellular MT content as quantified by the silver saturation method. This conclusion is supported by cell-free experiments in which the oxidation of rabbit MT-I by a xanthine/xanthine–oxidase system could be reversed by its subsequent reduction with 2-ME. The data presented provide direct evidence of the involvement of MT in scavenging superoxide radicals in living cells. Key words: macrophages, metallothionein, oxidative burst. Correspondence: V Albergoni, Department of Biology, University of Padova, via U. Bassi 58/B 35131 Padova, Italy. Email: biopd09@civ.bio.unipd.it Received 17 November 2000; accepted 30 January 2001.