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Free radicals are usually active species which have unpair electron (S) in molecules or Atomic groups. Of the free radicals, O2- and .OH could easily be produced in mammalian cells, by oxidation and reduction cycle catalyzed by fravoproteins and by iron + H2O2 reaction, respectively. Other free radicals would also be produced in mammalian cell, such as amino acid radicals, semiquinone radicals and flavine radicals etc. In general, free radicals cause cell injury though membrane lipid peroxidation and DNA strand cleavage and some other mechanisms.

The following species; superoxide (O2-.), hydrogen peroxide (H2O2), hydroxyl radical (.OH) and singlet oxygen (1O2), are generally called as reactive oxygen species (ROS). These species have been suggested to play important roles in various diseases caused by oxygen toxicity such as ischemia, carcinogenesis, inflammation, diabetes and aging. During the past two decades, considerable interests have been focused on chemical and biological research of ROS. We have also reported about the research results on ROS, which can be classified as following below; 1) chemical reactivities of O2-., 2) formation and toxicity of 1O2, 3) chemical reactivities of .OH, 4) enzyme mechanism of xanthine oxidase, 5) development of the compounds which induce the formation of O2-. and H2O2 in living cells and 6) development of superoxide dismutase mimics. These studies are reviewed from the standpoint of both chemical and biological interests.

The hydroxyl radicals (HO.) spin adduct of 5,5'-dimethyl-1-pyrroline-N-oxide (DMPO) was obtained in the hypoxanthine-xanthine oxidase (HX-X.O.) system in the presence of isolated islet cells by using electron spin resonance spectroscopy. There was a significant increase in the concentration of DMPO-OH spin adduct dependent on the increase in a number of islet cells. In the absence of islet cell, the DMPO-OOH spin adduct formed by superoxide together with a small amount of DMPO-OH spin adduct was detected in HX-X.O. system. These results suggest that HO. is greatly produced in the HX-X.O. system in the presence of islet cells. The generation of HO. was partially inhibited by the addition of superoxide dismutase or catalase and completely by the combined use of both enzymes. Iron-chelator, diethylenetriaminepentaacetic acid and iron-binding protein, apotransferrin also inhibited the generation of HO., suggesting a possible participation of a trace iron in the islet cells. The inhibition of insulin release from islet cell, as well as the amount of DMPO-OH spin adduct, was dependent on the concentration of X.O. These results indicate that the HO. is produced by the site-specific action with islet cells in HX-X.O. system, suggesting a possible involvement of HO. in the oxygen damages of islet cells.

We investigated the effect of recombinant human tumor necrosis factor (rhTNF) on hydroxyl radical production by established cell lines in vitro, and its implication in the killing of tumor cells by rhTNF. During incubation of TNF sensitive mouse tumorigenic fibroblast L-M cells in the presence of rhTNF, hydroxyl radical production detected by the evolution of methane gas from dimethyl sulfoxide increased gradually, at 18 hours reached 1.8 times of that in the absence of rhTNF. This increase of hydroxyl radical production was dependent on the concentration of rhTNF. The addition of iron chelator 2,2'-bipyridine which inhibits iron catalized Fenton reaction and inhibits hydroxyl radical generation, suppressed both the increase of hydroxyl radical production and the cytotoxicity induced by rhTNF. The increase of hydroxyl radical production stimulated by rhTNF was also detected in TNF sensitive human myosarcoma derived KYM cells as well as in L-M cells, but no change was detected in TNF insensitive human embryonic lung fibroblast HEL cells. These results suggest that rhTNF induced increase of hydroxyl radical production may play a significant role in the mechanism of tumor cell killing by rhTNF.