Inhibition of superoxide anion production in macrophages by anti-inflammatory drugs.

The inhibition by anti-inflammatory drugs of the production of Superoxide anions (O₂^?) by isolated guinea pig macrophages was studied spectrophotometrically using NADH and lactate dehydrogenase. id₅₀ values were: 4 × 10^?7M (diclophenac sodium), 1 × 10^?6M (oxyphenbutazone), 1 × 10^?5M (indomethacin), 4 × 10^?5M (phenylbutazone), 7 × 10^?5M (mefenamic acid), 8 × 10^?5 M (flufenamic acid), 8 × 10^?5M (colchicine), 3 × 10^?4M (aspirin), 3 × 10^?4M (benzydamine), 10^?3M < (dexamethasone) and 10^?3M < (gold sodium thiomalate). They seemed to block the cell membrane-associated mechanism to produce Superoxide anions, since most of them did not abolish the generation of superoxide anions from the xanthine oxidase plus hypoxanthine system. Cytochalasin B, pyrogallol, ascorbate, NEM, l-epinephrine and chlorpromazine also inhibited, the production of Superoxide anion, but many non anti-inflammatory drugs were ineffective. This technique was evaluated as a screening method in vitro for nonsteroidal anti-inflammatory drugs.     

Effects of borneol on the level of DNA damage induced in primary rat hepatocytes and testicular cells by hydrogen peroxide

The aim of this paper was to evaluate genotoxic effects of borneol and its ability to change DNA-damaging effects of H₂O₂ in rat hepatocytes and testicular cells. Both in vitro and ex vivo approaches were used in the case of hepatocytes. Testicular cells were tested only ex vivo, i.e. shortly after isolation from rats supplemented by borneol. Cytotoxicity of borneol increased in in vitro conditions in a concentration-dependent manner and it was associated with DNA-damaging effects at toxic concentrations. While non-toxic concentrations of borneol applied in vitro protected cells against H₂O₂-induced DNA damage and interfered only partly with rejoining of H₂O₂-induced DNA strand breaks, cytotoxic concentrations of borneol manifested synergy with H₂O₂, i.e. enhanced DNA-damaging effects of H₂O₂. On the other side, borneol given to rats in drinking water decreased the level of DNA damage induced by H₂O₂ in both hepatocytes and testicular cells. Our results show that though at higher concentrations (2-h treatment with >2 mM borneol >0.3084 mg/ml) borneol acts cytotoxically and genotoxically on primary hepatocytes cultured in vitro, if given to rats during 7 days in a daily concentration of 17.14 or 34.28 mg/kg it reduces genotoxicity of H₂O₂ in both hepatocytes and testicular cells.     

Limitation of the MTT and XTT assays for measuring cell viability due to superoxide formation induced by nano-scale TiO2

The reduction of the tetrazolium salts, MTT and XTT, is used to estimate cell viability and proliferation. However, superoxide can also reduce tetrazolium salts to produce the absorbant formazan end products. Evidence indicates that nano-TiO₂ induces superoxide formation in different mammalian cells. Therefore, studies investigating the cytological effects of nano-TiO₂ may encounter misleading results when using MTT/XTT to measure viability or proliferation. In this study, cell viabilities of Chinese hamster ovary cells were assayed using MTT, XTT and the trypan blue exclusion assay following exposure to nano-TiO₂. In comparison to the trypan blue exclusion assay, the MTT and XTT assays inaccurately predicted cell toxicity or overestimated cell viability respectively. XTT, in particular, appears more sensitive to superoxide than MTT. The reduction rate of XTT is 1.5 times that of MTT and SOD inhibition of XTT is less effective than that of MTT, indicating that XTT is more reactive with than MTT. Therefore, using XTT or MTT for measuring cell viability or proliferation may yield inaccurate results when conditions in cultured cell increase superoxide formation.