Francisco et al. Increase of reactive oxygen species by desferrioxamine during experimental Chagas’ disease Experimental animals comprised 48 male Swiss mice, each approximately 30 days old. Throughout the 35-day period of the study, animals received a commercial diet in pellet form (Nuvilab CR1) together with sterilized water ad libitum. Animals were distributed randomly into four equal groups (n = 12) as follows: control animals (NINT group) were not infected with T. cruzi and were not treated with DFO; group INT animals were infected with T. cruzi but were not treated with DFO; group NIT animals were not infected with T. cruzi but were treated with DFO; and group IT animals were infected with T. cruzi and treated with DFO. Animals belonging to the treatment groups received daily doses of DFO (5 mg; 0.05 ml) by intraperitoneal (IP) injection for 14 days prior to infection and for a further 21 days post-infection (dpi). Experimental animals in the NINT and INT groups were submitted to the same conditions of stress as the treated animals by daily administration of sterile water (0.05 ml) by IP injection. On day 14 of the study period, the 24 animals comprising IT and INT groups were infected by IP injection of 500 blood-stream forms of T. cruzi Y strain,12 which is considered to be partially resistant to chemotherapeutic treatment.5,13 Peripheral blood samples were collected from the orbital plexus of animals at 0, 7, 14 and 21 dpi. In each case, the serum was separated and stored frozen (–70ºC) until required for nitrate/nitrite and SOD analysis. In order to determine the levels of GT, TBARS and PC in hepatocytes, 12 animals in each group were sacrificed by cervical dislocation at 0, 7, 14 and 21 dpi, their livers were removed and immediately frozen to –70ºC and stored at this temperature until required for analysis. Assay of antioxidant enzymes SOD activity was quantified using Fluka (St Louis, MO, USA) SOD determination kit (catalogue #19160) based on the capacity of the antioxidant enzyme to inhibit the reduction of 2-(4-iodophenyl)-3- (4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt by superoxide anions (generated in situ by a xanthine/xanthine oxidase system) to yield the corresponding formazan dye (λmax 450 nm). Total glutathione (GT), i.e. reduced glutathione (GSH) and glutathione disulfide (GSSG), was assayed using a kinetic method (Sigma, St Louis, MO, USA; product code CS0260) in which GSH reduced 5,5’- dithiobis(2-nitrobenzoic acid) to 5-thio-2-nitrobenzoic acid (TNB) and GSSG, whilst the latter was recycled in situ by glutathione reductase and NADPH. TNB was determined spectrophotometrically at 412 nm. Assay of protein carbonyls The carbonyl moieties formed by the oxidation of protein by ROS were assayed using a conventional spectrophotometric procedure based on reaction with 2,4-dinitrophenyl hydrazine to form the corresponding hydrazone derivatives. Determination of serum PC concentration was carried out according to a published procedure,14 whilst total proteins were assayed according to the method of Lowry et al.15 PC was determined spectrophotometrically at 370 nm. Determination of lipid peroxidation Determination of TBARS concentration was based on the ability of the thiobarbituric acid to bind to oxidized lipids, this dosage was performed as described by Buege and Aust.16 TBARS were determined spectrophotometrically at 535 nm. Determination of serum nitrate/nitrite Levels of endogenously indirect production of NO were measured by nitrate/nitrite concentrations assessed by reducing serum nitrate to nitrite with nitrate reductase (1 U/ml) followed by the Griess reaction.17 Nitrite concentrations were determined by extrapolation from standard curves, constructed using various concentrations of sodium nitrite, and the results were expressed as micromolar. Nitrate/nitrite concentrations was measured by an ELISA plaque (Molecular Devices, E Max, USA) at 570 nm. Statistical analysis Between-group differences (NINT × NIT, INT × IT, NINT × INT and NIT × IT) in the levels of SOD, GT, nitrate/nitrite, TBARS and PC and between days after infection were analyzed by one-way analysis of variance and Tukey post-tests. In all cases, differences were considered statistically significant when P values were < 0.05. Results No differences between the experimental and control groups were observed in the levels of nitrate/nitrite at 0 and 7 dpi, PC at 0, 14 and 21 dpi and TBARS at 0 and 14 dpi. A significant increase in the level of TBARS in the INT group compared with the control (NINT group) was observed at 7 dpi (Fig. 1A), whilst at 21 dpi TBARS production in IT group animals was significantly increased compared with the NIT and INT groups (Fig. 1C). Figure 1B reveals that at 7 dpi, treatment with DFO had induced a significant increase in the levels of PC in the IT group compared with the NIT group. Significant increases in serum nitrate/nitrite levels were also observed at 21 dpi in animals of the IT Redox Report 2010 Vol 15 No 4 187