The cellular life-span of cultivated human skin epidermis keratinocytes NHEK-F was shown to be extended up to 150% of population doubling levels (PDLs) by repetitive addition with two autooxidation-resistant derivatives of ascorbic acid (Asc), Asc-2-O-phosphate (Asc2P), and Asc-2-O-alpha-glucoside (Asc2G), respectively, but to be not extended with Asc itself. In contrast, hydrogen peroxide (H(2)O(2)) as dilute as 20 microM which was non-cytotoxic to the keratinocytes, or at 60 microM being marginally cytotoxic achieved the cellular longevity, unexpectedly, up to 160 and 120% of PDLs, respectively, being regarded as a hormesis-like stimulatory effect. The lifespan-extended cells that were administered with Asc2P, Asc2G, or 20 microM H(2)O(2) were prevented from senescence-induced symptoms such as PDL-dependent enlargement of a cell size of 14.7 microm finally up to 17.4 microm upon Hayflick's limit-called loss of proliferation ability as estimated with a channelizer, and retained young cell morphological aspects such as thick and compact shape and intense attachment to the culture substratum even upon advanced PDLs, whereas other non-extended cells looked like thin or fibrous shape and large size upon lower PDLs. The PDL-dependent shortening of telomeric DNA of 11.5 kb finally down to 9.12-8.10 kb upon Hayflick's limit was observed in common for each additive-given cells, but was decelerated in the following order: 20 microM H(2)O(2) > Asc2P = Asc2G > 60 microM H(2)O(2) > Asc = no additive, being in accord with the order of cell longevity. Intracellular reactive oxygen species (ROS) was diminished by Asc2P, Asc2G or 20 microM H(2)O(2), but not significantly by Asc or 60 microM H(2)O(2) as estimated by fluorometry using the redox indicator dye CDCFH. There was no appreciable difference among NHEK keratinocytes that were administered with or without diverse additives in terms of telomerase activity per cell, which was 1.40 x 10(4)-4.48 x 10(4) times lower for the keratinocytes than for HeLa cells which were examined as the typical tumor cells. Thus longevity of the keratinocytes was suggested to be achieved by slowdown of age-dependent shortening of telomeric DNA rather than by telomerase; telomeres may suffer from less DNA lesions due to the continuous and thorough repression of intracellular ROS, which was realized either by pro-vitamin C such as Asc2P or Asc2G that exerted an antioxidant ability more persistent than Asc itself or by 20 microM H(2)O(2) which diminished intracellular ROS assumedly through a hormesis-like effect.

The ethanol extract from the fruit of Terminalia chebula (Combretaceae) exhibited significant inhibitory activity on oxidative stress and the age-dependent shortening of the telomeric DNA length. In the peroxidation model using t-BuOOH, the T. chebula extract showed a notable cytoprotective effect on the HEK-N/F cells with 60.5 +/- 3.8% at a concentration of 50 microg/ml. In addition, the T. chebula extract exhibited a significant cytoprotective effect against UVB-induced oxidative damage. The life-span of the HEK-N/F cells was elongated by 40% as a result of the continuous administration of 3 microg/ml of the T. chebula extract compared to that of the control. These observations were attributed to the inhibitory effect of the T. chebula extract on the age-dependent shortening of the telomere, length as shown by the Southern blots of the terminal restriction fragments (TRFs) of DNA extracted from subculture passages.

No abstract