To perform atomic absorption experiments
high purity water provided by a Milli-Q water purification system (Millipore, Bedford, MA, USA), nitric acid (Merck) and analytical solutions containing 1000 mg L−1 of Cu (CuCl2) (Titrisol®, Merck) were used. Calibrations curves were obtained by using reference solutions containing 0.5–5 mg L−1 of Cu2+ in 0.1% vol/vol HNO3. Direct analysis of cells was performed by weighing masses around 0.25 mg directly onto the graphite boat-type platform. A ZEEnit® 60 atomic absorption spectrometer (Analytik Jena AG, Jena, Germany) equipped with a manual HKI-272 datasheet solid sampling accessory, pyrolytic graphite tube atomizer and boat-type platform and hollow cathode lamp (wavelength = 216.5 nm, bandpass = 0.8 nm and lamp current = 4.0 mA) was used. A stainless steel
microspatula was used to transfer the samples to the pyrolytic boat-type platform. Microbalance Auto Balance AD-4 (Perkin-Elmer, Norwalk, USA) with a precision of 0.001 mg was used to weight samples. The heating program used for the direct determination of Cu in cells was adapted from a previous program developed by our group (step: temperature/°C, ramp/°C s− 1, hold/s): (drying: 180, 50, and 10), (pyrolysis: 1200, 100, and 15), (atomization: 2500, 2500, and 5) and (cleaning: 2600, Fulvestrant cost 1200, and 3) [49]. All experiments were repeated at least five times (except where otherwise stated) and data expressed as mean values and standard deviation. Differences between means were assessed by ANOVA with Bonferroni’s correction, and those with p values < 0.05 were considered significant. The aim of the study was to gain an insight into the mechanism by which the bicarbonate/carbon dioxide pair influences the generation of reactive species from hydrogen peroxide in the presence of different Cu(II) ions and complexes thereof. For this purpose, we have investigated the effect
on oxygen-derived radical formation of Cu(II) complexed with four different stable imine ligands [41], [42] and [43], cycling the metal between Glutamate dehydrogenase the 2+ and 1+ redox states, and with three low molecular weight peptide ligands known to form stable Cu(III) complexes in solution [44], [45], [46] and [47]. Assay of the rates of the copper-catalysed H2O2/HCO3− or H2O2-induced oxidation of DHR and NADH in vitro revealed that the generation of oxygen-derived radicals was much higher in the presence of Cu(II) sulphate than when Cu(II) imine complexes were present ( Fig. 2 and Fig. 3). This unexpected finding indicates that imine complexes generate lower levels of reactive oxygen species (ROS) than the free Cu(II) ion and Cu(II) peptide ligands, except Cu(GlyGlyHis). Such a result challenges the use of these complexes in cancer cell therapy to induce apoptosis in mammalian tumour cells in vitro on the basis of their facility to generate free radical and reactive species [35], [36], [37], [38] and [39].