Nutr Res Pract.  2007 Jun;1(2):105-112.

Ascorbic acid extends replicative life span of human embryonic fibroblast by reducing DNA and mitochondrial damages

• Hwang WS
• Park SH
• Kim HS
• Kang HJ
• Kim MJ
• Oh SJ
• Park JB
• Kim J
• Kim SC
• Lee JY

Abstract

Ascorbic acid has been reported to extend replicative life span of human embryonic fibroblast (HEF). Since the detailed molecular mechanism of this phenomenon has not been investigated, we attempted to elucidate. Continuous treatment of HEF cells with ascorbic acid (at 200 micrometer) from 40 population doubling (PD) increased maximum PD numbers by 18% and lowered SA-beta-gal positive staining, an aging marker, by 2.3 folds, indicating that ascorbic acid extends replicative life span of HEF cells. Ascorbic acid treatment lowered DCFH by about 7 folds and Rho123 by about 70%, suggesting that ascorbic acid dramatically decreased ROS formation. Ascorbic acid also increased aconitase activity, a marker of mitochondrial aging, by 41%, indicating that ascorbic acid treatment restores age-related decline of mitochondrial function. Cell cycle analysis by flow cytometry revealed that ascorbic acid treatment decreased G1 population up to 12%. Further western blot analysis showed that ascorbic acid treatment decreased levels of p53, phospho-p53 at ser 15, and p21, indicating that ascorbic acid relieved senescence-related G1 arrest. Analysis of AP (apurinic/apyrimidinic) sites showed that ascorbic acid treatment decreased AP site formation by 35%. We also tested the effect of hydrogen peroxide treatment, as an additional oxidative stress. Continuous treatment of 20 micrometer of hydrogen peroxide from PD 40 of HEF cells resulted in premature senescence due to increased ROS level, and increased AP sites. Taken together, the results suggest that ascorbic acid extends replicative life span of HEF cells by reducing mitochondrial and DNA damages through lowering cellular ROS.

Keyword

Ascorbic acid, human embryonic fibroblasts, replicative life span, mitochondrial damage, DNA damage; reactive oxygen species

MeSH Terms

Aconitate Hydratase
Aging
Ascorbic Acid*
Blotting, Western
Cell Cycle
DNA Damage
DNA*
Fibroblasts*
Flow Cytometry
Humans*
Hydrogen Peroxide
Oxidative Stress
Reactive Oxygen Species
Aconitate Hydratase
Ascorbic Acid
DNA
Hydrogen Peroxide
Reactive Oxygen Species

Figure

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  Fig. 1 The effect of ascorbic acid on growth and replicative life span of human embryonic fibroblast (HEF) cells. HEF cells were cultivated continuously from PD 40 with ascorbic acid and growth curve was prepared as described in "Materials and Methods". (A) The effect of ascorbic acid on replicative life span of HEF cells. The treatments of ascorbic acid are as follows; untreated (♦), 2 µM ascorbic acid (▾), 20 µM ascorbic acid (▴), 200 µM ascorbic acid (▪). (B) The effect of ascorbic acid and hydrogen peroxide on replicative life span of HEF cells. HEF cells were treated continuously with 20 µM hydrogen peroxide from PD 40. The treatments are as follows; 20 µM H2O2 (♦), 2 µM ascorbic acid and 20µM H2O2 (▾), 20 µM ascorbic acid and 20 µM H2O2 (▴), 200 µM ascorbic acid and 20 µM H2O2 (▪).

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  Fig. 2 The effect of ascorbic acid on SA-β-gal staining in HEF cells. (A) HEF cells were seeded at an initial density of 0.5 × 106/dish at PD 40 and cultivated continuously with ascorbic acid. SA-β-gal staining was performed (a) untreated old HEF cells (PD 64) (b) untreated young HEF cells (PD 30) (c) HEF cells treated with 200 µM ascorbic acid (PD 64) (d) HEF cells treated with 20 µM hydrogen peroxide (PD 56). (B) The percentage of SA-β-gal positive cells. SA-β-gal staining was performed on ascorbic acid-treated HEF cells (PD 64), untreated old HEF cells (PD 64), and H2O2-treated HEF cells (PD 58). The diagram was prepared from the staining result.

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  Fig. 3 Analysis of DCFH oxidation for measuring cellular ROS level in ascorbic acid-treated HEF cells. (A) Analysis of DCFH oxidation in ascorbic acid-treated HEF cells. HEF cells were treated with ascorbic acid beginning from PD 40. Oxidation of DCFH was determined at PD 64. (B) Analysis of DCFH oxidation in ascorbic acid/ H2O2-treated HEF cells. HEF cells were co-treated with 20 µM hydrogen peroxide and ascorbic acid beginning from PD 40.

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  Fig. 4 Analyses of Rho123 accumulation and aconitase activity as markers of mitochondrial function in ascorbic acid-treated HEF cells. HEF cells were treated with ascorbic acid beginning from PD 40. Ascorbic acidtreated HEF cells were harvested at PD 64 and used for analysis. (A) Analysis of Rho123 accumulation in ascorbic acid-treated HEF cells. Accumulation of Rho123 was determined as described in "Materials and Methods". (B) Analysis of Rho123 accumulation in ascorbic acid/ H2O2-treated HEF cells. HEF cells were co-treated with 20 µM hydrogen peroxide and ascorbic acid. Analysis of Rho123 accumulation was performed in old untreated HEF cells (PD 64) and H2O2-treated HEF cells (PD 58). (C) Analysis of aconitase activity in ascorbic acid-treated HEF cells. Cell extracts were promptly assayed for aconitase activity as described in "Materials and Methods".

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  Fig. 5 Cell cycle analysis of ascorbic acid-treated HEF cells. Ascorbic acid-treated HEF cells were stained with propidium iodide and cell cycle was determined by FACS analysis. Upper panel represents the results of FACS analysis and lower panel shows a plot prepared from above results. (A) Untreated old HEF cells (PD 62), (B) H2O2-treated old HEF cells (PD 56), (C) ascorbic acid-treated HEF cells (2 µM, PD 62), (D) ascorbic acid-treated HEF cells (20 µM, PD 62), (E) ascorbic acid-treated HEF cells (200 µM, PD 62).

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  Fig. 6 Western blot analysis of cell cycle marker proteins. Ascorbic acid-treated HEF cells were harvested and cell extracts were prepared. Western blot analysis was performed for cell extracts using specific antibodies against p21, p53, and phospho-p53 at ser 15. β-actin was used as a protein loading control. Lane 1; untreated old HEF cells (PD 54), lane 2; ascorbic acid-treated HEF cells (200 µM, PD 54), lane 3; ascorbic acid-treated HEF cells (200 µM, PD 64), lane 4; untreated old HEF cells (PD 64), lane 5; ascorbic acid-treated HEF cells (200 µM, PD 72).

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  Fig. 7 Analysis of AP sites in ascorbic acid-treated HEF cells. AP site were determined in ascorbic acid-treated HEF cells as described under "Materials and Methods". Samples were as follows; untreated-young HEF cells (PD 30), untreated-old HEF cells (PD 64), ascorbic acid-treated old HEF cells (2, 20, and 200 µM ascorbic acid, PD 64), and H2O2-treated HEF cells (PD 58).

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