Redox properties of metallothioneins (MTs) and Cu in the cytosol from Long-Evans Cinnamon (LEC) rat livers 13 weeks after birth were investigated. MTs from LEC rat livers contain 8 g atoms of Cu and 1 g atom of Zn per mole of protein (Cu(I)(8)-MTs). Titration of Cu(I)(8)-MTs with CuCl2 indicates that Cu(I)(8)-MTs were able to reduce further 2-g atoms of cupric ions per mole MTs as bound form. Hg2+-induced hydroxyl radical generation from Cu(I)(8)-MTs was demonstrated by ESR using the spin trap, 5,5-dimethyl-1-pyrroline N-oxide (DMPO). The intensity of DMPO-OH signal from Cu-loaded MTs was increased with the increasing number of Cu in MTs. The used cytosol fraction contained 1.37 mM total Cu and 5 mM DTNB titrable-SH groups has a potential to reduce 2 mM CuCl2. No ESR signal due to Cu2+ was also detected with LEC rat liver cytosol, whereas strong Cu2+ signal appeared by the addition of HgCl2. The rate constants for the reaction of Cu(I)(8)-MTs with superoxide and hydroxyl radicals were estimated to be 2 x 10(6) and greater than or equal to 10(12) M(-1)s(-1), respectively, from competition kinetics. Cu2+-catalyzed oxidation of DNA was strongly inhibited both in the presence of Cu-unsaturated MTs and GSH. The results suggest that Cu(I)(8)-MTs from LEC rat livers just before hepatitis still act as antioxidants. (C) 2001 Academic Press.