The kinetics of the effect of manganese supplementation on SOD2 activity in senescent human fibroblasts.

OBJECTIVE To investigate the effect of increasing Mn+2 concentrations on superoxide dismutase 2 (SOD2) activity in pre-senescent and senescent cultured fibroblasts, and to determine the Km Mn+2 values required to achieve maximal SOD2 activities in such cells. MATERIALS AND METHODS SOD2 activities, and superoxide anion (SOA) generation rates, were assayed in mitochondrial sonicates of young passage 5 fibroblasts sub-cultured in routine growth medium (MEM 1), and in an accurately identified senescent passage 20, 25 and 30 subcultures incubated with media containing supplemental Mn+2 increments equal to 60, 90, 120, 150 and 180 nM (MEM 2, 3, 4, 5 and 6 respectively). RESULTS Whereas SOD2 activity did not significantly change in any of the cells sub-cultured in MEM 1, the enzyme underwent progressive significant increases in early senescent passage 20 cells and senescent passage 25 and 30 cells. Such increases were relative to Mn+2 concentration and peaked in value in the senescent cells incubated with MEM 5 and MEM 6. Furthermore, whereas SOA generation rates underwent significant progressive increases in MEM 1-incubated senescent passage 20-30 cells, peaking in value at passage 30, the rates were gradually and significantly lowered in the cells incubated with MEM 2-MEM 6, and reached lowest values in those incubated with MEM 6 (p<0.001 for all comparisons). The computed Km values of Mn+2 with respect to SOD2 in senescent passage 20, 25 and 30 cells equalled 19.2, 39.6 and 54.4 nM respectively with corresponding SOD2 Vmax values of 37.6, 55.9 and 71.4 µmol/min/mg protein. CONCLUSIONS Senescent cells near the end of their replicative life span utilise more Mn+2 and achieve maximal SOD2 activities suggesting that the use of supplementary Mn+2 can help in combating oxidative stress.