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

H.K. Ghneim

Chair of Medical and Molecular Genetics Research, Department of Clinical Laboratory Sciences, College of Applied Medical Sciences, King Saud University, Riyadh, Saudi Arabia. hghneim@hotmail.com

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• Pharmacology

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.

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